Production of attenuated respiratory syncytial virus vaccines from cloned nucleotide sequences

ABSTRACT

Attenuated respiratory syncytial virus (RSV) and vaccine compositions thereof are produced by introducing specific mutations associated with attenuating phenotypes into wild-type or RSV which is incompletely attenuated by cold-passage or introduction of mutations which produce virus having a temperature sensitive (ts) or cold adapted (ca) phenotype. Alternatively, recombinant RSV and vaccine compositions thereof incorporate attenuating and other mutations specifying desired structural and or phenotypic characteristics in an infectious RSV. Recombinant RSV incorporate desired mutations specified by insertion, deletion, substitution or rearrangement of a selected nucleotide sequence, gene, or gene segment in an infectious RSV clone. The immune system of an individual is stimulated to induce protection against natural RSV infection, or multivalently against infection by RSV and another pathogen, such as PIV, by administration of attenuated, biologically derived or recombinant RSV.

RELATED APPLICATIONS

The present application claims the benefit of and is acontinuation-in-part of U.S. Provisional application Nos. 60/047,634,filed May 23, 1997, 60/046,141, filed May 9, 1997, and 60/021,773, filedJul. 15, 1996, each of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Human respiratory syncytial virus (RSV) outranks all other microbialpathogens as a cause of pneumonia and bronchiolitis in infants under oneyear of age. Virtually all children are infected by two years of age,and reinfection occurs with appreciable frequency in older children andyoung adults (Chanock et al., in Viral Infections of Humans, 3rd ed., A.S. Evans, ed., Plenum Press, N.Y. (1989)). RSV is responsible for morethan one in five pediatric hospital admissions due to respiratory tractdisease, and causes an estimated 91,000 hospitalizations and 4,500deaths yearly in the United States alone. Although most healthy adultsdo not have serious disease due to RSV infection, elderly patients andimmunocompromised individuals often suffer severe and possiblylife-threatening infections from this pathogen.

Despite decades of investigation to develop effective vaccine agentsagainst RSV, no safe and effective vaccine has yet been achieved toprevent the severe morbidity and significant mortality associated withRSV infection. Failure to develop successful vaccines relates in part tothe fact that small infants have diminished serum and secretory antibodyresponses to RSV antigens. Thus, these individuals suffer more severeinfections from RSV, whereas cumulative immunity appears to protectolder children and adults against more serious impacts of the virus. Oneantiviral compound, ribavarin, has shown promise in the treatment ofseverely infected infants, although there is no indication that itshortens the duration of hospitalization or diminishes the infant's needfor supportive therapy.

The mechanisms of immunity in RSV infection have recently come intofocus. Secretory antibodies appear to be most important in protectingthe upper respiratory tract, whereas high levels of serum antibodies arethought to have a major role in resistance to RSV infection in the lowerrespiratory tract. Purified human immunoglobulin containing a high titerof neutralizing antibodies to RSV may prove useful in some instances ofimmunotherapeutic approaches for serious lower respiratory tract diseasein infants and young children. Immune globulin preparations, however,suffer from several disadvantages, such as the possibility oftransmitting blood-borne viruses and difficulty and expense inpreparation and storage.

Formalin-inactivated virus vaccine was tested against RSV in themid-1960s, but failed to protect against RSV infection or disease, andin fact exacerbated symptoms during subsequent infection by the virus.(Kim et al., Am. J. Epidemiol., 89:422-434 (1969), Chin et al., Am J.Epidemiol., 89:449-463 (1969); Kapikian et al., Am. J. Epidemiol.,89:405-421 (1969)).

More recently, vaccine development for RSV has focused on attenuated RSVmutants. Friedewald et al., J. Amer. Med. Assoc. 204:690-694 (1968)reported a cold passaged mutant of RSV (cpRSV) which appeared to besufficiently attenuated to be a candidate vaccine. This mutant exhibiteda slight increased efficiency of growth at 26° C. compared to itswild-type parental virus, but its replication was neither temperaturesensitive nor significantly cold-adapted. The cold-passaged mutant,however, was attenuated for adults. Although satisfactorily attenuatedand immunogenic for infants and children who had been previouslyinfected with RSV (i.e., seropositive individuals), the cpRSV mutantretained a low level virulence for the upper respiratory tract ofseronegative infants.

Similarly, Gharpure et al., J. Virol. 3:414-421 (1969) reported theisolation of temperature sensitive RSV (tsRSV) mutants which also werepromising vaccine candidates. One mutant, ts-1, was evaluatedextensively in the laboratory and in volunteers. The mutant producedasymptomatic infection in adult volunteers and conferred resistance tochallenge with wild-type virus 45 days after immunization. Again, whileseropositive infants and children underwent asymptomatic infection,seronegative infants developed signs of rhinitis and other mildsymptoms. Furthermore, instability of the ts phenotype was detected,although virus exhibiting a partial or complete loss of temperaturesensitivity represented a small proportion of virus recoverable fromvaccinees, and was not associated with signs of disease other than mildrhinitis.

These and other studies revealed that certain cold-passaged andtemperature sensitive RSV strains were underattenuated and caused mildsymptoms of disease in some vaccinees, particularly seronegativeinfants, while others were overattenuated and failed to replicatesufficiently to elicit a protective immune response, (Wright et al.,Infect. Immun., 37:397-400 (1982)). Moreover, genetic instability ofcandidate vaccine mutants has resulted in loss of theirtemperature-sensitive phenotype, further hindering development ofeffective RSV vaccines. See generally, Hodes et al., Proc. Soc. Exp.Biol. Med. 145:1158-1164 (1974), McIntosh et al., Pediatr. Res.8:689-696 (1974), and Belshe et al., J. Med. Virol., 3:101-110 (1978).

Abandoning the attenuated RS virus vaccine approach, investigatorstested potential subunit vaccine candidates using purified RS virusenvelope glycoproteins from lysates of infected cells. The glycoproteinsinduced resistance to RS virus infection in the lungs of cotton rats,Walsh et al., J. Infect. Dis. 155:1198-1204 (1987), but the antibodieshad very weak neutralizing activity and immunization of rodents withpurified subunit vaccine led to disease potentiation (Murphy et al.,Vaccine 8:497-502 (1990)).

Vaccinia virus recombinant-based vaccines which express the F or Genvelope glycoprotein have also been explored. These recombinantsexpress RSV glycoproteins which are indistinguishable from the authenticviral counterpart, and small rodents infected intradermally with thevaccinia-RSV F and G recombinant viruses developed high levels ofspecific antibodies that neutralized viral infectivity. Indeed,infection of cotton rats with vaccinia-F recombinants stimulated almostcomplete resistance to replication of RSV in the lower respiratory tractand significant resistance in the upper tract. Olmsted et al., Proc.Natl. Acad. Sci. USA 83:7462-7466 (1986). However, immunization ofchimpanzees with vaccinia F and vaccinia G recombinant provided almostno protection against RSV challenge in the upper respiratory tract(Collins et al., Vaccine 8:164-168 (1990)) and inconsistent protectionin the lower respiratory tract (Crowe et al., Vaccine 11:1395-1404(1993).

The unfulfilled promises of attenuated RSV strains, subunit vaccines,and other strategies for RSV vaccine development underscores a need fornew methods to identify genetic targets for recombinant engineering ofnovel RSV vaccines, and to develop methods for manipulating recombinantRSV to incorporate genetic changes to yield new phenotypic properties inviable, attenuated RSV recombinants. However, manipulation of thegenomic RNA of RSV and other negative-sense RNA viruses has heretoforeproven difficult. Major obstacles in this regard include non-infectivityof naked genomic RNA of these viruses, poor viral growth in tissueculture, lengthy replication cycles, virion instability, a complexgenome, and a refractory organization of gene products.

Methods for direct genetic manipulation of nonsegmented, negativestranded RNA viruses have only recently begun to be developed (forreviews, see Conzelmann, J. Gen. Virol. 77:381-89 (1996); Palese et al.,Proc. Natl. Acad. Sci. U.S.A. 93:11354-58, (1996)). Successful rescuehas been achieved for infectious rabies virus, vesicular stomatitisvirus (VSV), measles virus, and Sendai virus from cDNA-encodedantigenomic RNA in the presence of the nucleocapsid N, phosphoprotein P,and large polymerase subunit L (Garcin et al., EMBO J. 14:6087-6094(1995); Lawson et al., Proc. Natl. Acad. Sci. U.S.A. 92:4477-81 (1995);Radecke et al., EMBO J. 14:5773-5784 (1995); Schnell et al., EMBO J.13:4195-203 (1994); Whelan et al., Proc. Natl. Acad. Sci. U.S.A.92:8388-92 (1995)). Successful rescue of RSV also requires an additionalprotein, the M2 ORF1 transcriptional elongation factor (Collins et al.,Proc. Natl. Acad. Sci. USA 92:11563-7 (1995)).

Rescue of infectious RSV has been complicated by a number of factors.Specifically, RSV possesses several properties which distinguish it andother members of the genus Pneumovirus from better characterizedparamyxoviruses of the genera Paramyxovirus, Rubulavirus andMorbillivirus. These differences include a greater number of mRNAs, anunusual gene order at the 3′ end of the genome, species-to-speciesvariability in the order of the glycoprotein and M2 genes, a greaterdiversity in intergenic regions, an attachment protein that exhibitsmucin-like characteristics, extensive strain-to-strain sequencediversity, and several proteins not found in other nonsegmented negativestranded RNA viruses.

In view of the foregoing, an urgent need exists in the art for tools andmethods to engineer safe and effective vaccines to alleviate the serioushealth problems attributable to RSV, particularly illnesses amonginfants and children. Quite surprisingly, the present inventionsatisfies these and other related needs.

SUMMARY OF THE INVENTION

The present invention provides novel methods and compositions fordesigning and producing isolated attenuated respiratory syncytial virus(RSV). RSV of the invention are either biologically derived fromwild-type or selected RSV mutant parental stocks or are recombinantlyengineered to incorporate phenotype-specific genetic changes. RSVdesigned and selected for vaccine use have at least two and sometimes atleast three attenuating mutations. In one embodiment, at least oneattenuating mutation occurs in the RSV polymerase gene and involves anucleotide substitution specifying an amino acid change in thepolymerase protein resulting in a temperature-sensitive (ts) phenotype.Exemplary biologically derived and recombinant RSV incorporate one ormore nucleotide substitutions in the large polymerase gene L resultingin an amino acid change at amino acid Phe₅₂₁, Gln₈₃₁, Met₁₁₆₉ orTyr₁₃₂₁, as exemplified by the changes, Leu for Phe₅₂₁, Leu for Gln₈₃₁,Val for Met₁₁₆₉, and Asn for Tyr₁₃₂₁. Alternately or additionally, RSVof the invention may comprise a nucleotide substitution in a differentRSV gene, e.g., in the M2 gene.

Attenuating mutations may be selected in coding portions of a RSV gene,or in a non-coding portion such as a cis-regulatory sequence. Exemplarynon-coding mutations include single or multiple base changes in a genestart sequence, as exemplified by a single or multiple base substitutionin the M2 gene start sequence at nucleotide 7605. Preferably, two orthree mutations are incorporated in a codon specifying an attenuatingmutation, e.g., in a codon specifying a ts mutation at amino acidPhe₅₂₁, Gln₈₃₁, Met₁₁₆₉, or Tyr₁₃₂₁, thereby decreasing any likelihoodof reversion from the ts phenotype.

In other embodiments of the invention, biologically derived orrecombinant RSV are selected or engineered to incorporate at least twoattenuating ts mutations, e.g., in the polymerase gene at amino acidPhe₅₂₁ and Met₁₁₆₉ as exemplified by the novel, biologically derivedvaccine strain cpts-530/1009, ATCC No. VR 2451, at Phe₅₂₁ and Tyr₁₃₂₁,as exemplified by the biologically derived strain cpts-530/1030, ATCCNo. VR 2455, at Gln₈₃₁, and the M2 gene start mutation at nt 7605, asexemplified by the biologically derived RSV strain cpts-248/404, ATCCNo. VR 2454, or at the M2 gene start mutation at nt 7605, and Phe₅₂₁ asexemplified by the recombinant RSV rA2cp/248/404/530.

Biologically derived RSV into which selected attenuating mutations canbe introduced include wild-type RSV, a host range-restrictedcold-passaged RSV, a cold adapted mutant of host range-restrictedcold-passaged RSV, or a temperature sensitive strain. For example, thecold-passaged respiratory syncytial virus can be the cpRSV strain,including cpRSV into which at least two temperature-sensitive mutationshave been introduced. The attenuated RSV virus can be subgroup A, asexemplified by cpts RSV 248 (ATCC VR 2450), cpts 248/404 (ATCC VR 2454),cpts 248/955 (ATCC VR 2453), cpts RSV 530 (ATCC VR 2452), cpts 530/1009(ATCC VR 2451), or cpts 530/1030 (ATCC VR 2455), or subgroup B, asexemplified by B-1 cp52/2B5 (ATCC VR 2542) or B-1 cp-23, each depositedunder the terms of the Budapest Treaty with the American Type CultureCollection (ATCC) of 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A.,and granted the above identified accession numbers.

In another aspect of the invention, an isolated infectious recombinantRSV is provided which is generated from a RSV genome or antigenome, anucleocapsid (N) protein, a nucleocapsid phosphoprotein (P), a large (L)polymerase protein, and an RNA polymerase elongation factor. The RNApolymerase elongation factor can be M2(ORF1) of RSV. The isolatedinfectious RSV can be a viral or subviral particle. The genome orantigenome can comprise a wild-type RSV sequence, a biologically derivedmutant sequence specifying an attenuated phenotype, or a recombinant RSVsequence encoding a wild-type RSV genome or antigenome or arecombinantly modified RSV sequence comprising an engineered mutation orcombination of mutations not found in any biologically derived RSVstrain.

The infectious RSV clone can incorporate coding or non-coding nucleotidesequences from any RSV or RSV-like virus, e.g., human, bovine or, murineRSV (pneumonia virus of mice, or avian RSV (turkey rhinotracheitisvirus, or from another virus, e.g., parainfluenza virus (PIV). Inexemplary aspects, the recombinant RSV comprises a chimera of a humanRSV genomic or antigenomic sequence recombinantly joined with aheterologous RSV sequence. Exemplary heterologous sequences include RSVsequences from one human RSV strain combined with sequences from adifferent human RSV strain (e.g., a chimera of sequences from two ormore strains selected from cpts RSV 248, cpts 248/404, cpts 248/955,cpts RSV 530, cpts 530/1009, or cpts 530/1030), or subgroup (e.g. acombination of RSV subgroup A and subgroup B sequences), from a nonhumanRSV, or from another virus such as PIV.

In one embodiment of the invention, recombinant RSV are provided whereinthe genome or antigenome is recombinantly altered, e.g., compared to awild-type or biologically derived mutant sequence. Mutationsincorporated within recombinantly altered RSV clones may be selectedbased on their ability to alter expression and/or function of a selectedRSV protein, yielding a desired phenotypic change, or for a variety ofother purposes. Desired phenotypic changes include, e.g., changes inviral growth in culture, temperature sensitivity, plaque size,attenuation, and immunogenicity. For example, a polynucleotide sequenceencoding the genome or antigenome can be modified by a nucleotideinsertion, rearrangement, deletion or substitution to specifyattenuation, temperature-sensitivity, cold-adaptation, small plaquesize, host range restriction, alteration in gene expression, or a changein an immunogenic epitope.

In one aspect, recombinant RSV are provided wherein at least oneattenuating mutation occurs in the RSV polymerase gene L and involves anucleotide substitution specifying a (ts) phenotype. Exemplary RSVclones incorporate a nucleotide substitution resulting in an amino acidchange in the polymerase gene at Phe₅₂₁, Gln₈₃₁, Met₁₁₆₉ or Tyr₁₃₂₁.Preferably, two or three mutations are incorporated in a codonspecifying an attenuating mutation. Other exemplary RSV clonesincorporate at least two attenuating ts mutations.

Mutations occurring in biologically derived, attenuated RSV can beintroduced individually or in combination into a full-length RSV clone,and the phenotypes of rescued recombinant viruses containing theintroduced mutations can be readily determined. In exemplaryembodiments, amino acid changes displayed by attenuated,biologically-derived viruses over a wild-type RSV, for example changesexhibited by cold-passaged RSV (cpRSV) or a further attenuated RSVstrain such as a temperature-sensitive derivative of cpRSV (cptsRSV),are incorporated within recombinant RSV clones. These changes from awild-type or biologically derived mutant RSV sequence specify desiredcharacteristics in the resultant clones, e.g., an attenuated or furtherattenuated phenotype. These changes are preferably introduced intorecombinant virus using two or three nucleotide changes compared to acorresponding wild type or biologically derived mutant sequence, whichhas the effect of stabilizing the mutation against genetic reversion.

The present invention also provides recombinant RSV having multiple,phenotype-specific mutations introduced in selected combinations intothe genome or antigenome of an infectious clone. This process, coupledwith evaluation of phenotype, provides mutant recombinant RSV havingsuch desired characteristics as attenuation, temperature sensitivity,cold-adaptation, small plaque size, host range restriction, etc.Mutations thus identified are compiled into a “menu” and introduced invarious combinations to calibrate a vaccine virus to a selected level ofattenuation, immunogenicity and stability. In preferred embodiments, theinvention provides for supplementation of one or more mutations adoptedfrom biologically derived RSV, e.g., cp and ts mutations, withadditional types of mutations involving the same or different genes.Target genes for mutation in this context include the attachment (G)protein, fusion (F) protein, small hydrophobic (SH), RNA binding protein(N), phosphoprotein (P), the large polymerase protein (L), thetranscription elongation factor (M2), M2 ORF2, the matrix (M) protein,and two nonstructural proteins, NS1 and NS2. Each of these proteins canbe selectively deleted, substituted or rearranged, in whole or in part,alone or in combination with other desired modifications, to achievenovel RSV recombinants. In one aspect, the SH gene, is deleted to yielda recombinant RSV having novel phenotypic characteristics, includingenhanced growth in vitro and/or attenuation in vivo. In a relatedaspect, this gene deletion, or another selected, non-essential gene orgene segment deletion, such as a NS1 or NS2 gene deletion is combined ina recombinant RSV with one or more mutations specifying an attenuatedphenotype, e.g., a mutation adopted directly (or in modified form, e.g.,by introducing multiple nucleotide changes in a codon specifying themutation) from a biologically derived attenuated RSV mutant. Forexample, the SH gene or NS2 gene may be deleted in combination with oneor more cp and/or ts mutations adopted from cpts248/404, cpts530/1009,cpts530/1030, or another selected mutant RSV strain, to yield arecombinant RSV having increased yield of virus, enhanced attenuation,and genetic resistance to reversion from an attenuated phenotype, due tothe combined effects of the different mutations.

In addition, a variety of other genetic alterations can be produced in arecombinant RSV genome or antigenome for incorporation into infectiousrecombinant RSV, alone or together with one or more attenuating pointmutations adopted from a biologically derived mutant RSV. Heterologousgenes (e.g. from different RSV strains or subgroups or non-RSV sources)may be inserted in whole or in part, the order of genes changed, geneoverlap removed, the RSV genome promoter replaced with its antigenomecounterpart, portions of genes removed or substituted, and even entiregenes deleted. Different or additional modifications in the sequence canbe made to facilitate manipulations, such as the insertion of uniquerestriction sites in various intergenic regions (e.g., a unique Stulsite between the G and F genes) or elsewhere. Nontranslated genesequences can be removed to increase capacity for inserting foreignsequences.

In exemplary embodiments, individual genes, gene segments, or single ormultiple nucleotides of one RSV may be substituted by counterpartsequence(s) from a heterologous RSV or other source. For example, aselected, heterologous gene segment, such as one encoding a cytoplasmictail, transmembrane domain or ectodomain, an epitopic site or region, abinding site or region, an active site or region containing an activesite, etc., of a selected protein from one RSV, can be substituted for acounterpart gene segment in another RSV to yield novel recombinants, forexample recombinants expressing a chimeric protein having a cytoplasmictail and/or transmembrane domain of one RSV fused to an ectodomain ofanother RSV. In one embodiment, F and/or G protective antigens of oneRSV strain or subgroup are substituted into an RSV clone of a differentstrain or subgroup to produce a recombinant virus capable of stimulatinga cross-protective immune response against both strains or subgroups inan immunized host. In additional aspects, a chimeric RSV clone having amutation involving alteration of a gene or gene segment, e.g., asubstituted, heterologous F and/or G gene or gene segment, is furthermodified by introducing one or more attenuating ts or cp point mutationsadopted from a biologically derived mutant RSV strain, e.g.,cpts248/404, cpts530/1009, or cpts530/1030. In yet additional aspects,one or more human RSV coding or non-coding polynucleotides aresubstituted with a counterpart sequence from bovine or murine RSV, aloneor in combination with one or more selected cp or ts mutations, to yieldnovel attenuated vaccine strains. In one embodiment, a chimericbovine-human RSV incorporates a substitution of the human RSV NP gene orgene segment with a counterpart bovine NP gene or gene segment, whichchimera can optionally be constructed to incorporate a SH gene deletion,one or more cp or ts point mutations, or various combinations of theseand other mutations disclosed herein.

Alternatively, the polynucleotide molecule encoding the RSV genome orantigenome can be modified to encode non-RSV sequences, e.g., acytokine, a T-helper epitope, a restriction site marker, or a protein ofa microbial pathogen (e.g., virus, bacterium or fungus) capable ofeliciting a protective immune response in the intended host.

In another aspect of the invention, novel methods are provided forintroducing the aforementioned structural and phenotypic changes into arecombinant RSV to yield infectious, attenuated vaccine viruses. In oneembodiment, an expression vector is provided which comprises an isolatedpolynucleotide molecule encoding a RSV genome or antigenome. Alsoprovided is the same or different expression vector comprising one ormore isolated polynucleotide molecules encoding N, P, L and RNApolymerase elongation factor proteins. The vector(s) is/are coexpressedin a cell or cell-free lysate, thereby producing an infectious RSVparticle. The RSV genome or antigenome and the N, P, L and RNApolymerase elongation factor proteins can be coexpressed by the same ordifferent expression vectors. In some instances the N, P, L and RNApolymerase elongation factor proteins are each encoded on differentexpression vectors. The polynucleotide molecule encoding the RSV genomeor antigenome is from a human, bovine or murine RSV sequence, or can bea chimera of different RSV or non-RSV sequences, for example apolynucleotide containing sequences from a subgroup A RSV operablyjoined with sequences from a subgroupe B RSV, or containing RSVsequences operably joined with PIV sequences. The RSV genome orantigenome can be modified from a wild-type or biologically derivedmutant RSV strain by insertion, rearrangement, deletion or substitutionof one or more nucleotides, including point mutations, site-specificnucleotide changes, and changes involving entire genes or gene segments.These alterations typically specify a selected phenotypic change in theresulting recombinant RSV, such as a phenotypic change that results inattenuation, temperature-sensitivity, cold-adaptation, small plaquesize, host range restriction, alteration in gene expression, or a changein an immunogenic epitope.

In other embodiments the invention provides a cell or cell-free lysatecontaining an expression vector which comprises an isolatedpolynucleotide molecule encoding a RSV genome or antigenome and anexpression vector which comprises one or more isolated polynucleotidemolecules that encodes N, P, L and RNA polymerase elongation factorproteins of RSV. Upon expression the genome or antigenome and N, P, L,and RNA polymerase elongation factor proteins combine to produce aninfectious RSV particle, such as viral or subviral particle.

The attenuated RSV of the invention is capable of eliciting a protectiveimmune response in an infected human host, yet is sufficientlyattenuated so as to not cause unacceptable symptoms of severerespiratory disease in the immunized host. The attenuated virus may bepresent in a cell culture supernatant, isolated from the culture, orpartially or completely purified. The virus may also be lyophilized, andcan be combined with a variety of other components for storage ordelivery to a host, as desired.

The invention further provides novel vaccines comprising aphysiologically acceptable carrier and/or adjuvant and an isolatedattenuated RSV strain. In one embodiment, the vaccine is comprised ofinfectious RSV having at least two and sometimes three or moreattenuating mutations, at least one of which results in atemperature-sensitive substitution at amino acid Phe₅₂₁, Gln₈₃₁,Met₁₁₆₉, or Tyr₁₃₂₁ in the respiratory syncytial virus polymerase gene.In other embodiments, the vaccine comprises a recombinant, attenuatedRSV. The vaccine can be formulated in a dose of 10³ to 10⁶ PFU ofattenuated virus. The vaccine may comprise attenuated virus of eitherantigenic subgroup A or B, or virus of both subgroups A and B can becombined in vaccine formulations for more comprehensive coverage againstprevalent RSV infections.

In other aspects the invention provides a method for stimulating theimmune system of an individual for more comprehensive coverage againstprevalent RSV or to induce protection against RSV. The method comprisesadministering a vaccine formulated in an immunologically sufficientamount of an attenuated respiratory syncytial virus of the invention. Inone embodiment, the vaccine comprises infectious RSV having multipleattenuating mutations, at least one of which specifies a ts substitutionat amino acid Phe₅₂₁, Gln₈₃₁, Met₁₁₆₉, or Tyr₁₃₂₁ in the L gene. Inalternate embodiments, the vaccine comprises a recombinant, attenuatedRSV. The virus will typically be administered with an acceptable carrierand/or adjuvant. The method may include administering attenuated virusof subgroup A and/or subgroup B to the individual, in an amount of 10³to 10⁶ PFU to the upper respiratory tract by spray, droplet, aerosol, orthe like. Generally the attenuated virus is administered intranasally toan individual seronegative for antibodies to said virus, or to anindividual who possesses transplacentally acquired maternal anti-RSVantibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph demonstrating the substantially complete correlationbetween the replication of a series of subgroup A respiratory syncytialviruses in the lungs of mice with their replication in the chimpanzee.

FIGS. 2 and 3 show the construction of cDNA encoding RSV antigenome RNA,where FIG. 2 shows the structures of the cDNA and the encoded antigenomeRNA (not to scale). For the purposes of the present Figures, and in allsubsequent Examples hereinbelow, the specific cDNAs and viruses usedwere of strain A2 of subgroup A RSV. The diagram of the antigenomeincludes the following features: the 5′-terminal nonviral G tripletcontributed by the T7 promoter, the four sequence markers at positions1099 (which adds one nt to the length), 1139, 5611, and 7559 (numberingreferring to the first base of the new restriction site), the ribozymeand tandem T7 terminators, and the single nonviral 3′-phosphorylated Uresidue contributed to the 3′ end by ribozyme cleavage (the site ofcleavage is indicated with an arrow). Note that the nonviral 5′-GGG and3′-U residues are not included in length values given here andthereafter for the antigenome. However, the nucleotide insertion atposition 1099 is included, and thus the numbering for cDNA-derivedantigenome is one nucleotide greater downstream of this position thanfor biologically derived antigenome. The 5′ to 3′ positive-sensesequence of D46 (the genome itself being negative-sense) is depicted inSEQ ID NO: 1, where the nucleotide at position four can be either C orG. Also note that the sequence positions assigned to restriction sitesin this Figure and throughout are intended as a descriptive guide and donot alone define all of the nucleotides involved. The length valuesassigned to restriction fragments here and throughout also aredescriptive, since length assignments may vary based on such factors assticky ends left following digestion. Cloned cDNA segments representingin aggregate the complete antigenome are also shown. The box illustratesthe removal of the BamHI site from the plasmid vector, a modificationthat facilitated assembly: the naturally occurring BamHI-SalI fragment(the BamHI site is shown in the top line in positive sense, underlined)was replaced with a PCR-generated BglII-SalI fragment (the BglII site isshown in the bottom line, underlined; its 4-nt sticky end, shown initalics, is compatible with that of BamHI). This resulted in a single ntchange (middle line, underlined) which was silent at the amino acidlevel. FIG. 3 shows the sequence markers contained in the cDNA-encodedantigenome RNA, where sequences are positive sense and numbered relativeto the first nt of the leader region complement as 1; identities betweenstrains A2 and 18537, representing RSV subgroups A and B, respectively,are indicated with dots; sequences representing restriction sites in thecDNA are underlined; gene-start (GS) and gene-end (GE) transcriptionsignals are boxed; the initiation codon of the N translational openreading frame at position 1141 is italicized, and the restriction sitesare shown underneath each sequence. In the top sequence, a single Cresidue was inserted at position 1099 to create an AflII site in theNS2-N intergenic region, and the AG at positions 1139 and 1140immediately upstream of the N translational open reading frame werereplaced with CC to create a new NcoI site. In the middle sequence,substitution of G and U at positions 5612 and 5616, respectively,created a new StuI site in the G-F intergenic region. In the bottomsequence, a C replacement at position 7560 created a new SphI site inthe F-M2 intergenic region.

FIG. 4 illustrates structures of cDNAs (approximately to scale) involvedin the insertion of mutations, assembly of complete antigenomeconstructs, and recovery of recombinant virus. Four types of mutationswere inserted into the pUC118- or pUC119-borne cDNA subclones shown inthe bottom row, namely six silent restriction sites in the L gene(underlined over the D53 diagram on the top), two HEK changes in the Fgene (H), five cp changes (cp), and the mutations specific to thevarious biological mutagenesis steps: 248, 404, 530, 1009, and 1030 (asindicated). The mutagenized subclones were inserted into the D50(representing the RSV antigenome from the leader to the beginning of theM2-L overlap with the T7 promoter immediately upstream of the leader) orD39 (representing the RSV antigenome from the M2-L overlap to thetrailer with the ribozyme and T7 terminators immediately downstream ofthe trailer) intermediate plasmids shown in the middle row. Theappropriate D50 and D39 were assembled into full-length D53 antigenomecDNA as shown on the top row (RTT indicates the location of thehammer-head ribozyme followed by two T7 transcription terminators).

FIG. 5 provides maps of six mutant antigenome cDNAs which were used torecover recombinant RSV. The ts phenotypes of the recombinants aresummarized on the right of the figure.

FIG. 6 shows construction of D46/1024CAT cDNA encoding an RSV antigenomecontaining the CAT ORF flanked by RSV transcription signals (not toscale, RSV-specific segments are shown as filled boxes and CAT sequenceas an open box). The source of the CAT gene transcription cassette wasRSV-CAT minigenome cDNA 6196 (diagram at top). The RSV-CAT minigenomecontains the leader region, GS and GE signals, noncoding (NC) RSV genesequences, and the CAT ORF, with XmaI restriction endonuclease sitespreceding the GS signal and following the GE signal. The nucleotidelengths of these elements are indicated, and the sequences(positive-sense) surrounding the XmaI sites are shown above the diagram.A 8-nucleotide XmaI linker was inserted into StuI site of the parentalplasmid D46 to construct the plasmid D46/1024. D46 is the completeantigenome cDNA and is equivalent to D53; the difference in nomenclatureis to denote that these represent two different preparations. TheXma-XmaI fragment of the plasmid 6196 was inserted into the plasmidD46/1024 to construct the plasmid D46/1024CAT. The RNA encoded by theD46 cDNA is shown at the bottom, including the three 5′-terminalnonviral G residues contributed by the T7 promoter and the 3′-terminalphosphorylated U residue contributed by cleavage of the hammerheadribozyme; the nucleotide lengths given for the antigenome do not includethese nonviral nucleotides. The L gene is drawn offset to indicate thegene overlap.

FIG. 7 is a diagram (not to scale) of the parental wild type D46 plasmidencoding an RSV antigenome (top), and the D46/6368 derivative in whichthe SH gene has been deleted (bottom). The RSV genes are shown as openrectangles with the GS and GE transcription signals shown as filledboxes on the upstream and downstream ends, respectively. The T7 phagepromoter (left) and hammerhead ribozyme and T7 terminators used togenerate the 3′ end of the RNA transcript (right) are shown as smallopen boxes. The ScaI and PacI fragment of D46 was replaced with a shortsynthetic fragment, resulting in D46/6368. The sequence flanking the SHgene in D46, and the sequence of the engineered region in D46/6368, areeach shown framed in a box over the respective plasmid map. The sequenceof the ScaI-PacI fragment in D46, and its replacement in D46/6368, areshown in bold and demarcated with arrows facing upward. The M GE, SH GS,SH GE and G GS sites are indicated with overlining. The new M-Gintergenic region in D46/6368 is labeled 65 in the diagram at the bottomto indicate its nucleotide length. The positive-sense T7 transcript ofthe SH-minus D46/6368 construct is illustrated at the bottom; the three5′-terminal nonviral G residues contributed by the T7 promoter and the3′-terminal U residue are shown (Collins, et al. 1995, Proc. Natl. Acad.Sci. USA 92:11563-11567, incorporated herein by reference). Thesenonviral nucleotides are not included in length measurements.

FIG. 8 provides results of RT-PCR analysis of total intracellular RNAfrom cells infected with the D46 wild type or D46/6368 SH-minus virus toconfirm the deletion in the SH locus. RT was performed with apositive-sense primer that anneals upstream of the SH gene, and the PCRemployed in addition a negative-sense primer that anneals downstream ofthe SH gene. Lanes: (1 and 5) markers consisting of the 1 Kb DNA ladder(Life Technologies, Gaithersburg, Md.); (2) D46/6368 RNA subjected toRT-PCR; (3) D46 RNA subjected to RT-PCR; (4) D46/6368 RNA subjected toPCR alone. PCR products were electrophoresed on a 2.5% agarose gel andstained with ethidium bromide. Nucleotides lengths of some marker DNAfragments are shown to the right.

FIG. 9 shows Northern blot hybridization of RNAs encoded by the D46 wildtype and D46/6368 SH-minus virus. Total intracellular RNA was isolatedfrom infected cells and subjected to oligo(dT) chromatography without aprior denaturation step, conditions under which the selected. RNA alsoincludes genomic RNA due to sandwich hybridization. RNAs wereelectrophoresed on formaldehyde-agarose gels and blotted ontonitrocellulose membrane. Replicate blots were hybridized individuallywith [³²P]-labeled DNA probes of the M, SH, G, F, M2, or L genes, asindicated. Lanes: (1) D46/6368 RNA; (2) D46 RNA; (3) uninfected HEp-2cell RNA. Positions of the genomic RNA (gen.), mRNAs (large letters) andread-through transcripts (small letters) are shown on the left. Thepositions of the read-through transcripts P-M and M-G (M probe)coincide, as well as the positions of G-F and F-M2 transcripts (Fprobe). The positions of the 0.24-9.5 kb RNA ladder molecular weightmarkers (Life Technologies), which was run in parallel and visualized byhybridization with [³²P]-labeled DNA of phage lambda, are shown on theright.

FIG. 10 shows SDS-PAGE of [³⁵S]-labeled RSV proteins synthesized inHEp-2 cells infected with the D46 wild type or D46/6368 SH-minus virus.Proteins were subjected to immunoprecipitation with antiserum raisedagainst purified virions and analyzed by electrophoresis in pre-castgradient 4%-20% Tris-glycine gels (Novex, San Diego, Calif.). Positionsof viral proteins are indicated to the left; positions and molecularmasses (in kilodaltons) of marker proteins (Kaleidoscope PrestainedStandards, Bio-Rad, Richmond, Calif.), are shown to the right.

FIGS. 11-13 provide growth curves for D46 wild type and D46/6368SH-minus viruses in HEp-2 cells (FIG. 11), 293 cells (FIG. 12), andAGMK-21 cells (FIG. 13). Triplicate cell monolayers in 25-cm² cultureflasks were infected with 2 PFU per cell of either virus, and incubatedat 37° C. Aliquots were taken at indicated time points, stored at −70°C., and titrated in parallel by plaque assay with antibody staining.Each point shown is the average titer of three infected cell monolayers.

FIGS. 14 and 15 show kinetics of virus replication in the upper (FIG.14) and lower (FIG. 15) respiratory tract of mice inoculatedintranasally with the D46 wild type virus, D46/6368 SH-minus virus, orthe biologically-derived cpts248/404 virus. Mice in groups of 24 wereinoculated intranasally with 10⁶ PFU of the indicated virus. Six micefrom each group were sacrificed on the indicated day and the nasalturbinates and lung tissues were removed and homogenized, and levels ofinfectious virus were determined by plaque assay on individual specimensand mean log₁₀ titers were determined.

FIG. 16 shows a comparison of the transcription products and gene orderof SH-minus virus compared to its wild type counterpart. The upper panelsummarizes an analysis of the amounts of certain mRNAs produced by theSH-minus virus compared with the wild type parent recombinant virus.Intracellular mRNAs were isolated from cells infected with the SH-minusor wild type virus and analyzed by Northern blot hybridization withgene-specific probes. The amount of hybridized radioactivity wasquantitated, and the relative abundance of each individual mRNA producedby the SH-minus virus versus its wild type parent is shown. The lowerpanel shows the gene order of the wild type virus from the M gene(position 5 in the gene order) to the L gene (position 10). This iscompared to that of the SH-minus virus, in which the positions in thegene order of the G, F, M2 and L genes are altered due to deletion ofthe SH gene.

FIG. 17 depicts the D46 antigenome plasmid which was modified bydeletion of the SH gene in such a way as to not insert any heterologoussequence into the recombinant virus. The sequence flanking the SH genedepicted at the top. The MGE, M-SH intergenic (IG), SH GS, SH GE andSH-G IG sequences are shown. The area which was removed by the deletionis underlined, with the deletion points indicated with upward pointingtriangles. The antigenome resulting from this deletion is D46/6340.

FIG. 18 depicts the introduction of tandem translation stop codons intothe translational open reading frame (ORF) encoding the NS2 protein.Plasmid D13 contains the left end of the antigenome cDNA, including theT7 promoter (shaded box), the leader region, and the NS1, NS2, N, P, Mand SH genes. Only the cDNA insert of D13 is shown. The AatII-AflIIfragment containing the T7 promoter and NS1 and NS2 genes was subclonedinto a pGem vector, and site-directed mutagenesis was used to modify theNS2 ORF in the region illustrated by the sequence. The wild typesequence of codons 18 to 26 is shown (the encoded amino acids areindicated below), and the three nucleotides above are the threesubstitutions which were made to introduce two termination codons (ter)and an XhoI site (underlined) as a marker. The resulting cDNA andsubsequent recovered virus are referred to as NS2-knockout (KO).

FIG. 19 compares production of infectious virus by wild type RSV (D53)versus NS2-knockout RSV in HEp-2 cells. Triplicate monolayers wereinfected with either virus at an input moi of three pfu/cell, andsamples were taken at the indicated intervals and quantitated by plaqueassay and immunostaining.

FIG. 20 depicts alteration of gene-end (GE) signals of the NS1 and NS2genes. The cDNA insert of plasmid D13, representing the left hand end ofthe antigenome cDNA from the T7 promoter (shaded) to the PacI site atposition 4623, is shown. The AatI-AflII fragment containing the T7promoter and the NS1 and NS2 genes was subcloned into a pGem vector. Itwas modified by site-directed mutagenesis simultaneously at two sites,namely the NS1 and NS2 GE signals were each modified to be identical tothat found in nature for the N gene. The sequences of the wild type NS1and NS2 GE signals are shown (and identified by sequence positionrelative to the complete antigenome sequence), and the nucleotidesubstitutions are shown above the line. The dash in the wild typesequence of the NS2 GE signal indicates that the mutation increased thelength of the GE signal by one nucleotide.

FIG. 21 depicts the deletion of the polynucleotide sequence encoding theNS1 protein. The left hand part of the D13 cDNA is shown at the bottom:D13 contains the left hand part of the antigenome cDNA, from the leaderto the end of the SH gene, with the T7 promoter immediately upstream ofthe leader. The sequence on either side of the deletion point (upwardarrow) is shown on top. The deletion spans from immediately before thetranslational start site of the NS1 ORF to immediately before that ofthe NS2 ORF. Thus, it has the effect of fusing the NS1 GS and upstreamnoncoding region to the NS2 ORF. This precludes the disruption of anycis-acting sequence elements which might extend into the NS1 gene due toits leader-proximal location.

FIG. 22 depicts the deletion of the polynucleotide sequence encoding theNS2 mRNA. As described above, the left hand part of the D13 cDNA isshown along with the sequence on either side of the deletion point(upward arrow). The deletion spans from immediately downstream of theNS1 gene to immediately downstream of the NS2 gene. Thus, the sequenceencoding the NS2 mRNA has been deleted in its entirety, but no othersequence has been disrupted. The resulting cDNA and subsequent recoveredrecombinant virus are referred to as ΔNS2.

FIG. 23 depicts the ablation of the translational start site for thesecreted form of the G protein. The 298-amino acid G protein is shown asan open rectangle with the signal-anchor sequence filled in. The aminoacid sequence for positions 45 to 53 is shown overhead to illustrate twonucleotide substitutions which change amino acid 48 from methionine toisoleucine and amino acid 49 from isoleucine to valine. The formermutation eliminates the translational start site for the secreted form.The two mutations also create an MfeI site, which provides a convenientmethod for detecting the mutation. The resulting cDNA and subsequentrecovered virus are referred to as M48I (methionine-48 toisoleucine-48).

FIG. 24 shows the results of a comparison of production of infectiousvirus by wild type RSV (D53) versus that of two isolates of recoveredD53/M481 membrane G mutant virus.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides biologically derived and recombinant RSVsuitable for vaccine use in humans. Attenuated RSV described herein areproduced by introducing and/or combining specific attenuating mutationsinto incompletely attenuated strains of RSV, such as a temperaturesensitive (ts) or cold-passaged (cp) RS virus, or into wild-type virus,e.g. RSV strain A2. The mutations in biologically derived RSV may occurnaturally or may be introduced into selected RSV strains by well knownmutagenesis or similar procedures. For example, incompletely attenuatedparental RSV strains can be produced by chemical mutagenesis duringvirus growth in cell cultures to which a chemical mutagen has beenadded, by selection of virus that has been subjected to passage atsuboptimal temperatures in order to introduce growth restrictionmutations, or by selection of a mutagenized virus that produces smallplaques (sp) in cell culture, as generally described herein and in U.S.Ser. No. 08/327,263, incorporated herein by reference.

By “biologically derived RSV” is meant any RSV not produced byrecombinant means. Thus, biologically derived RSV include naturallyoccurring RSV of all subgroups and strains, including, e.g., naturallyoccurring RSV having a wild-type genomic sequence and RSV having genomicvariations from a reference wild-type RSV sequence, e.g., RSV having amutation specifying an attenuated phenotype. Likewise, biologicallyderived RSV include RSV mutants derived from a parental RSV strain by,inter alia, artificial mutagenesis and selection procedures.

To produce a satisfactorily attenuated RS virus of the invention,mutations are preferably introduced into a parental viral strain whichhas been incompletely or partially attenuated, such as the ts-1 or ts-4mutant, or cpRSV. For virus of subgroup A, the incompletely attenuatedparental virus is preferably ts-1 or ts-1NG or cpRSV, which are widelyknown mutants of the A2 strain of subgroup A, or derivatives orsubclones thereof. In other embodiments the specific mutations which areassociated with attenuated phenotypes are identified and introduced intostrains suitable for vaccine use. The strains into which the specificattenuating mutations are introduced can be wild-type virus or can bealready partially attenuated, in which case the additional mutation(s)further attenuate the strain, e.g., to a desired level of restrictedreplication in a mammalian host while retaining sufficientimmunogenicity to confer protection in vaccinees.

Partially attenuated mutants of the subgroup A or B virus can beproduced by biologically cloning wild-type virus in an acceptable cellsubstrate and developing cold-passaged mutants thereof, subjecting thevirus to chemical mutagenesis to produce ts mutants, or selecting smallplaque or similar phenotypic mutants thereof (see, e.g., Murphy et al.,International Publication WO 93/21310, incorporated herein byreference). For virus of subgroup B, an exemplary, partially attenuatedparental virus is cp 52/2B5, which is a mutant of the B1 strain ofsubgroup B. The various selection techniques may be combined to producepartially attenuated mutants from non-attenuated subgroup A or B strainswhich are useful for further derivatization as described herein.Further, mutations specifying attenuated phenotypes may be introducedindividually or in combination in incompletely attenuated subgroup A orB virus to produce vaccine virus having multiple, defined attenuatingmutations that confer a desired level of attenuation and immunogenicityin vaccinees.

Once a desired, partially attenuated parental strain is selected,further attenuation sufficient to produce a vaccine acceptable for usein humans according to the present invention may be accomplished inseveral ways as described herein. For example, a cpRSV mutant can befurther mutagenized in several ways. In one embodiment the procedureinvolves subjecting the partially attenuated virus to passage in cellculture at progressively lower, attenuating temperatures. For example,whereas wild-type virus is typically cultivated at about 34-37° C., thepartially attenuated mutants are produced by passage in cell cultures(e.g., primary bovine kidney cells) at suboptimal temperatures, e.g.,20-26° C. Thus, in one method of the present invention the cp mutant orother partially attenuated strain, e.g., ts-1 or sp, is adapted toefficient growth at a lower temperature by passage in MRC-5 or Verocells, down to a temperature of about 20-24° C., preferably 20-22° C.This selection of mutant RS virus during cold-passage substantiallyeliminates any residual virulence in the derivative strains as comparedto the partially attenuated parent. Alternatively, specific mutationscan be introduced into a recombinant version via mutagenesis or othermethods to further mutagenize a cp mutant, e.g., mutations responsiblefor ts, sp or ca.

In one embodiment of the invention the incompletely attenuated RSVstrains are subjected to chemical mutagenesis to introduce ts mutationsor, in the case of viruses which are already ts, additional ts mutationssufficient to confer increased stability of the ts phenotype on theattenuated derivative. Means for the introduction of ts mutations intoRS virus include replication of the virus in the presence of a mutagensuch as 5-fluorouridine or 5-fluorouracil in a concentration of about10⁻³ to 10⁻⁵ M, preferably about 10⁻⁴ M, exposure of virus tonitrosoguanidine at a concentration of about 100 μg/ml, according to thegeneral procedure described in, e.g., Gharpure et al., J. Virol.3:414-421 (1969) and Richardson et al., J. Med. Virol. 3:91-100 (1978),or genetic introduction of specific ts mutations. Other chemicalmutagens can also be used. Attenuation can result from a ts mutation inalmost any RS virus gene, and as identified herein ts mutations aretypically associated with the polymerase (L) gene.

The level of temperature sensitivity of replication in exemplaryattenuated RSV of the invention is determined by comparing itsreplication at a permissive temperature with that at several restrictivetemperatures. The lowest temperature at which the replication of thevirus is reduced 100-fold or more in comparison with its replication atthe permissive temperature is termed the shutoff temperature. Inexperimental animals and humans, both the replication and virulence ofRSV correlate with the mutant's shutoff temperature. Replication ofmutants with a shutoff temperature of 39° C. is moderately restricted,whereas mutants with a shutoff of 38° C. replicate less well andsymptoms of illness are mainly restricted to the upper respiratorytract. A virus with a shutoff temperature of 35 to 37° C. will typicallybe fully attenuated in humans. Thus, the attenuated RSV of the inventionwhich is ts will have a shutoff temperature in the range of about 35 to39° C., and preferably from 35 to 38° C. The addition of a ts mutationto a partially attenuated strain produces multiply attenuated virususeful in the vaccine compositions of the present invention.

A number of attenuated RSV strains as candidate vaccines for intranasaladministration have been developed using multiple rounds of chemicalmutagenesis to introduce multiple mutations into a virus which hadalready been attenuated during cold-passage (e.g., Connors et al.,Virology 208: 478-484 (1995); Crowe et al., Vaccine 12: 691-699 (1994);and Crowe et al., Vaccine 12: 783-790 (1994), incorporated herein byreference). Evaluation in rodents, chimpanzees, adults and infantsindicate that certain of these candidate vaccine strains are relativelystable genetically, are highly immunogenic, and may be satisfactorilyattenuated. Nucleotide sequence analysis of some of these attenuatedviruses, as exemplified hereinbelow, indicates that each level ofincreased attenuation is associated with specific nucleotide and aminoacid substitutions. The present invention provides the ability todistinguish between silent incidental mutations versus those responsiblefor phenotype differences by introducing the mutations, separately andin various combinations, into the genome or antigenome of infectious RSVclones. This process coupled with evaluation of phenotypecharacteristics of parental and derivative virus identifies mutationsresponsible for such desired characteristics as attenuation, temperaturesensitivity, cold-adaptation, small plaque size, host range restriction,etc. Mutations thus identified are compiled into a “menu” and are thenbe introduced as desired, singly or in combination, to calibrate avaccine virus to an appropriate level of attenuation, immunogenicity,genetic resistance to reversion from an attenuated phenotype, etc., asdesired.

The specific mutations which have been introduced into RSV by serialrandom chemical mutagenesis to create further attenuated RSV having a tsphenotype are identified by sequence analysis and comparison to theparental background, e.g., a cpRSV background. In an exemplaryembodiment, substitution within the polymerase (L) gene of nucleotide10,989 (A changed to T, resulting in gln to leu amino acid substitutionat amino acid residue 831) to create cptsRSV 248, or at nucleotide10,060 (C changed to A, resulting in a phe to leu amino acidsubstitution at amino acid residue 521) yielded cptsRSV 530. Thesecombined attenuating mutations specify a greater attenuation phenotypethan determined for the parental cpRSV. cptsRSV 248 has a shutofftemperature of 38° C., it is attenuated, immunogenic, and fullyprotective against wild-type RSV challenge in seronegative chimpanzees,and it is more stable genetically during replication in vivo thanpreviously evaluated RSV mutants. The attributes of genetic resistanceto phenotypic reversion, and restriction of replication in vivo, alsoindicate that this exemplary strain is a desirable parental strain intowhich additional mutations are introduced.

In another exemplary embodiment, the cptsRSV 248 mutant was subjected tochemical mutagenesis, and a series of further attenuated mutants, e.g.,having increased temperature sensitivity or small plaque phenotypecharacteristics over the parental strain, were produced. One suchmutant, designated cptsRSV 248/404, is characterized by a reducedshutoff temperature of 36° C. which is the consequence of an additionalmutation at nucleotide 7605 (T changed to C), with this being in anoncoding highly conserved gene start cis-acting regulatory sequence andnot resulting in an amino acid change.

In another embodiment of the invention, RSV mutants are produced havingtwo or more attenuating mutations. For example, the multiply attenuatedvirus cptsRSV 530, which has a shutoff temperature of 39° C., wassubjected to chemical mutagenesis to introduce one or more additionalmutations specifying an attenuating phenotype in a non-attenuatedbackground. In one example, the clone cptsRSV 530/1009 was isolated anddetermined to have a shutoff temperature of 36° C. due to a singlenucleotide substitution at 12,002 (A changed to G, resulting in a met toval amino acid substitution at amino acid residue 1169), also in thepolymerase (L) gene.

The present invention also provides infectious RSV produced byrecombinant methods, e.g., from cDNA. In one embodiment, infectious RSVis produced by the intracellular coexpression of a cDNA that encodes theRSV genome or antigenome RNA, together with those viral proteinsnecessary to generate a transcribing, replicating nucleocapsid,preferably one or more sequences that encode major nucleocapsid (N)protein, nucleocapsid phosphoprotein (P), large (L) polymerase protein,and a transcriptional elongation factor M2 ORF1 protein.

The ability to produce infectious RSV from cDNA permits the introductionof specific engineered changes, including site specific attenuatingmutations and a broad spectrum of other recombinant changes, into thegenome of a recombinant virus to produce safe, effective RSV vaccines.

The infectious, recombinant RSV of the invention are employed herein foridentification of specific mutation(s) in biologically derived,attenuated RSV strains, for example mutations which specify ts, ca, attand other phenotypes. Accordingly, the invention permits incorporationof biologically derived mutations, along with a broad range of otherdesired changes, into recombinant RSV vaccine strains. For example, thecapability of producing virus from cDNA allows for incorporation ofmutations occurring in attenuated RSV vaccine candidates to beintroduced, individually or in various selected combinations, into afull-length cDNA clone, and the phenotypes of rescued recombinantviruses containing the introduced mutations to be readily determined. Inexemplary embodiments, amino acid changes identified in attenuated,biologically-derived viruses, for example in a cold-passaged RSV(cpRSV), or in a further attenuated strain derived therefrom, such as atemperature-sensitive derivative of cpRSV (cptsRSV), are incorporatedwithin recombinant RSV clones. These changes from a wild-type orbiologically derived mutant RSV sequence specify desired characteristicsin the resultant clones, e.g., an attenuated or further attenuatedphenotype compared to a wild-type or incompletely attenuated parentalRSV phenotype.

By identifying and incorporating specific, biologically derivedmutations associated with desired phenotypes, e.g., a cp or tsphenotype, into infectious RSV clones, the invention provides for other,site-specific modifications at, or within the region of, the identifiedmutation. Whereas most attenuating mutations produced in biologicallyderived RSV are single nucleotide changes, other site specific mutationscan also be incorporated by recombinant techniques into biologicallyderived or recombinant RSV. As used herein, site-specific mutationsinclude insertions, substitutions, deletions or rearrangements of from 1to 3, up to about 5-15 or more altered nucleotides (e.g., altered from awild-type RSV sequence, from a sequence of a selected mutant RSV strain,or from a parent recombinant RSV clone subjected to mutagenesis). Suchsite-specific mutations may be incorporated at, or within the region of,a selected, biologically derived point mutation. Alternatively, themutations can be introduced in various other contexts within an RSVclone, for example at or near a cis-acting regulatory sequence ornucleotide sequence encoding a protein active site, binding site,immunogenic epitope, etc. Site-specific RSV mutants typically retain adesired attenuating phenotype, but may exhibit substantially alteredphenotypic characteristics unrelated to attenuation, e.g., enhanced orbroadened immunogenicity, or improved growth. Further examples ofdesired, site-specific mutants include recombinant RSV designed toincorporate additional, stabilizing nucleotide mutations in a codonspecifying an attenuating point mutation. Where possible, two or morenucleotide substitutions are introduced at codons that specifyattenuating amino acid changes in a parent mutant or recombinant RSVclone, yielding a biologically derived or recombinant RSV having geneticresistance to reversion from an attenuated phenotype. In otherembodiments, site-specific nucleotide substitutions, additions,deletions or rearrangements are introduced upstream (N-terminaldirection) or downstream (C-terminal direction), e.g, from 1 to 3, 5-10and up to 15 nucleotides or more 5′ or 3′, relative to a targetednucleotide position, e.g., to construct or ablate an existing cis-actingregulatory element.

In addition to single and multiple point mutations and site-specificmutations, changes to recombinant RSV disclosed herein includedeletions, insertions, substitutions or rearrangements of whole genes orgene segments. These mutations affect small numbers of bases (e.g., from15-30 bases, up to 35-50 bases or more), or large blocks of nucleotides(e.g., 50-100, 100-300, 300-500, 500-1,000 bases) depending upon thenature of the change (i.e., a small number of bases may be changed toinsert or ablate an immunogenic epitope or change a small gene segment,whereas large block(s) of bases are involved when genes or large genesegments are added, substituted, deleted or rearranged.

In additional aspects, the invention provides for supplementation ofmutations adopted from biologically derived RSV, e.g., cp and tsmutations which occur mainly in the L gene, with additional types ofmutations involving the same or different genes in a recombinant RSVclone. RSV encodes ten mRNAs and ten or eleven proteins. Three of theseare transmembrane surface proteins, namely the attachment G protein,fusion F protein involved in penetration, and small hydrophobic SHprotein. G and F are the major viral neutralization and protectiveantigens. Four additional proteins are associated with the viralnucleocapsid, namely the RNA binding protein N, the phosphoprotein P,the large polymerase protein L, and the transcription elongation factorM2 ORF1. The matrix M protein is part of the inner virion and probablymediates association between the nucleocapsid and the envelope. Finally,there are two nonstructural proteins, NS1 and NS2, of unknown function.These proteins can be selectively altered in terms of its expressionlevel, or can be added deleted, substituted or rearranged, in whole orin part, alone or in combination with other desired modifications, in arecombinant RSV to obtain novel infectious RSV clones.

Thus, in addition to, or in combination with, attenuating mutationsadopted from biologically derived RSV mutants, the present inventionalso provides entirely new methods of attenuation based on recombinantengineering of infectious RSV clones. In accordance with this aspect ofthe invention, a variety of alterations can now be produced in anisolated polynucleotide sequence encoding the RSV genome or antigenomefor incorporation into infectious recombinant RSV. More specifically, toachieve desired structural and phenotypic changes in recombinant RSV,the invention allows for introduction of modifications which delete,substitute, introduce, or rearrange a selected nucleotide or pluralityof nucleotides from a parent RSV sequence, as well as mutations whichdelete, substitute, introduce or rearrange whole gene(s) or genesegment(s), within an infectious RSV clone.

Desired modifications of infectious recombinant RSV are typicallyselected to specify a desired phenotypic change, e.g., a change in viralgrowth, temperature sensitivity, ability to elicit a host immuneresponse, attenuation, etc. These changes can be brought about by, e.g.,mutagenesis of a parent RSV clone to ablate, introduce or rearrange aspecific gene(s) or gene region(s) (e.g., a gene segment that encodes aprotein structural domain, such as a cytoplasmic, transmembrane orextracellular domain, an immunogenic epitope, binding region, activesite, etc.). Genes of interest in this regard include all of the genesof the RSV genome: 3′-NS1-NS2-N-P-M-SH-G-F-M2-L-5′, as well asheterologous genes from other RSV, other viruses and a variety of othernon-RSV sources as indicated herein. Also provided are modificationswhich simply alter or ablate expression of a selected gene, e.g., byintroducing a termination codon within a selected RSV coding sequence;changing the position of an RSV gene relative to an operably linkedpromoter; introducing an upstream start codon to alter rates ofexpression; modifying (e.g., by changing position, altering an existingsequence, or substituting an existing sequence with a heterologoussequence) GS and/or GE transcription signals to alter phenotype (e.g.,growth, temperature restrictions on transcription, etc.); and variousother deletions, substitutions, additions and rearrangements thatspecify quantitative or qualitative changes in viral replication,transcription of selected gene(s), or translation of selectedprotein(s).

As described herein, cDNA-based methods are used to construct a seriesof recombinant viruses offering improved characteristics of attenuationand immunogenicity for use as vaccine agents. Among desired phenotypicchanges in this context are resistance to reversion from an attenuatedphenotype, improvements in attenuation in culture or in a selected hostenvironment, immunogenic characteristics (e.g., as determined byenhancement, or diminution, of an elicited immune response),upregulation or downregulation of transcription and/or translation ofselected viral products, etc. Foreign genes may be inserted in whole orin part, the order of genes changed, gene overlap removed, the RSVgenome promoter replaced with its antigenome counterpart, portions ofgenes (e.g., the cytoplasmic tails of glycoprotein genes) added (e.g.,to duplicate an existing sequence or incorporate a heterologoussequence), removed or substituted, and even entire genes deleted.

In one aspect of the invention, a selected gene segment, such as oneencoding a selected protein or protein region (e.g., a cytoplasmic tail,transmembrane domain or ectodomain, an epitopic site or region, abinding site or region, an active site or region containing an activesite, etc.) from one RSV, can be substituted for a counterpart genesegment from the same or different RSV or other source, to yield novelrecombinants having desired phenotypic changes compared to wild-type orparent RSV strains. For example, recombinants of this type may express achimeric protein having a cytoplasmic tail and/or transmembrane domainof one RSV fused to an ectodomain of another RSV. Other exemplaryrecombinants of this type express duplicate protein regions, such asduplicate immunogenic regions. As used herein, “counterpart” genes, genesegments, proteins or protein regions, are typically from heterologoussources (e.g., from different RSV genes, or representing the same (i.e.,homologous or allelic) gene or gene segment in different RSV strains).Typical counterparts selected in this context share gross structuralfeatures, e.g., each counterpart may encode a comparable structural“domain,” such as a cytoplasmic domain, transmembrane domain,ectodomain, binding site or region, epitopic site or region, etc.Counterpart domains and their encoding gene segments embrace anassemblage of species having a range of size and amino acid (ornucleotide) sequence variations, which range is defined by a commonbiological activity among the domain or gene segment variants. Forexample, two selected protein domains encoded by counterpart genesegments within the invention may share substantially the samequalitative activity, such as providing a membrane spanning function, aspecific binding activity, an immunological recognition site, etc. Moretypically, a specific biological activity shared between counterparts,e.g., between selected protein segments or proteins, will besubstantially similar in quantitative terms, i.e., they will not vary inrespective quantitative activity profiles by more than 30%, preferablyby no more than 20%, more preferably by no more than 5-10%.

Counterpart genes and gene segments, as well as other polynucleotidesinvolved in producing recombinant RSV within the invention, preferablyshare substantial sequence identity with a selected polynucleotidereference sequence, e.g., with another selected counterpart sequence. Asused herein, a “reference sequence” is a defined sequence used as abasis for a sequence comparison, for example, a segment of a full-lengthcDNA or gene, or a complete cDNA or gene sequence. Generally, areference sequence is at least 20 nucleotides in length, frequently atleast 25 nucleotides in length, and often at least 50 nucleotides inlength. Since two polynucleotides may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide sequence) that issimilar between the two polynucleotides, and (2) may further comprise asequence that is divergent between the two polynucleotides, sequencecomparisons between two (or more) polynucleotides are typicallyperformed by comparing sequences of the two polynucleotides over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window”, as used herein, refers to aconceptual segment of at least 20 contiguous nucleotide positionswherein a polynucleotide sequence may be compared to a referencesequence of at least 20 contiguous nucleotides and wherein the portionof the polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) of 20 percent or less as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. Optimal alignment ofsequences for aligning a comparison window may be conducted by the localhomology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981),by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443 (1970), by the search for similarity method of Pearson & Lipman,Proc. Natl. Acad. Sci. USA 85:2444 (1988) (each of which is incorporatedby reference), by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.,incorporated herein by reference), or by inspection, and the bestalignment (i.e., resulting in the highest percentage of sequencesimilarity over the comparison window) generated by the various methodsis selected. The term “sequence identity” means that two polynucleotidesequences are identical (i.e., on a nucleotide-by-nucleotide basis) overthe window of comparison. The term “percentage of sequence identity” iscalculated by comparing two optimally aligned sequences over the windowof comparison, determining the number of positions at which theidentical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity. The terms “substantialidentity” as used herein denotes a characteristic of a polynucleotidesequence, wherein the polynucleotide comprises a sequence that has atleast 85 percent sequence identity, preferably at least 90 to 95 percentsequence identity, more usually at least 99 percent sequence identity ascompared to a reference sequence over a comparison window of at least 20nucleotide positions, frequently over a window of at least 25-50nucleotides, wherein the percentage of sequence identity is calculatedby comparing the reference sequence to the polynucleotide sequence whichmay include deletions or additions which total 20 percent or less of thereference sequence over the window of comparison. The reference sequencemay be a subset of a larger sequence.

In addition to these polynucleotide sequence relationships, proteins andprotein regions encoded by recombinant RSV of the invention are alsotypically selected to have conservative relationships, i.e., to havesubstantial sequence identity or sequence similarity, with selectedreference polypeptides. As applied to polypeptides, the term “sequenceidentity” means peptides share identical amino acids at correspondingpositions. The term “sequence similarity” means peptides have identicalor similar amino acids (i.e., conservative substitutions) atcorresponding positions. The term “substantial sequence identity” meansthat two peptide sequences, when optimally aligned, such as by theprograms GAP or BESTFIT using default gap weights, share at least 80percent sequence identity, preferably at least 90 percent sequenceidentity, more preferably at least 95 percent sequence identity or more(e.g., 99 percent sequence identity). The term “substantial similarity”means that two peptide sequences share corresponding percentages ofsequence similarity. Preferably, residue positions which are notidentical differ by conservative amino acid substitutions. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. Stereoisomers (e.g., D-amino acids) of the twentyconventional amino acids, unnatural amino acids such asα,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, andother unconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4-hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,ω-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). Moreover, amino acids may be modified byglycosylation, phosphorylation and the like.

In alternative aspects of the invention, the infectious RSV producedfrom cDNA-expressed genome or antigenome can be any of the RSV orRSV-like strains, e.g., human, bovine, murine, etc., or of anypneumovirus, e.g., pneumonia virus of mice or turkey rhinotracheitisvirus. To engender a protective immune response, the RSV strain may beone which is endogenous to the subject being immunized, such as humanRSV being used to immunize humans. The genome or antigenome ofendogenous RSV can be modified, however, to express RSV genes or genesegments from a combination of different sources, e.g., a combination ofgenes or gene segments from different RSV species, subgroups, orstrains, or from an RSV and another respiratory pathogen such as PIV(see, e.g., Hoffman et al., J. Virol. 71:4272-4277 (1997); Durbin etal., Virology (1997) (In Press); Murphy et al., U.S. Patent ApplicationSer. No. 60/047,575 entitled PRODUCTION OF INFECTIOUS PARAINFLUENZAVIRUS FROM CLONED NUCLEOTIDE SEQUENCES, filed May 23, 1997, incorporatedherein by reference, and the following plasmids for producing infectiousPIV clones: p3/7(131) (ATCC 97990); p3/7(131)2G (ATCC 97889); andp218(131) (ATCC 97991); each deposited under the terms of the BudapestTreaty with the American Type Culture Collection (ATCC) of 12301Parklawn Drive, Rockville, Md. 20852, U.S.A., and granted the aboveidentified accession numbers.

In certain embodiments of the invention, recombinant RSV are providedwherein individual internal genes of a human RSV are replaced with,e.g., a bovine murine or other RSV counterpart, or with a counterpart orforeign gene from another respiratory pathogen such as PIV.Substitutions, deletions, etc. of RSV genes or gene segments in thiscontext can include part or all of one or more of the NS1, NS2, N, P, M,SH, M2(ORF1), M2(ORF2) and L genes, or non-immunogenic parts of the Gand F genes. Also, human RSV cis-acting sequences, such as promoter ortranscription signals, can be replaced with, e.g., their bovine RSVcounterpart. Reciprocally, means are provided to generate liveattenuated bovine RSV by inserting human attenuating genes or cis-actingsequences into a bovine RSV genome or antigenome background.

Recombinant RSV bearing heterologous genes or cis-acting elements areselected for host range restriction and other desired phenotypesfavorable for vaccine use. In exemplary embodiments, bovine RSVsequences are selected for introduction into human RSV based onavailable descriptions of bovine RSV structure and function, as providedin, e.g., Pastey et al., J. Gen. Viol. 76:193-197 (1993); Pastey et al.,Virus Res. 29:195-202 (1993); Zamora et al., J. Gen. Virol. 73:737-741(1992); Mallipeddi et al., J. Gen. Virol. 74:2001-2004 (1993);Mallipeddi et al., J. Gen. Virol. 73:2441-2444 (1992); and Zamora etal., Virus Res. 24:115-121 (1992), each of which is incorporated hereinby reference, and in accordance with the methods and compositionsdisclosed herein. In other embodiments, mutations of interest forintroduction within recombinant RSV are modeled after a tissueculture-adapted nonpathogenic strain of pneumonia virus of mice (themurine counterpart of human RSV) which lacks a cytoplasmic tail of the Gprotein (Randhawa et al., Virology 207:240-245 (1995)). Accordingly, inone aspect of the invention the cytoplasmic and/or transmembrane domainsof one or more of the human RSV glycoproteins, F, G and SH, are added,deleted, modified, or substituted with a heterologous, counterpartsequence (e.g., a sequence from a cytoplasmic, or transmembrane domainof a F, G, or SH protein of bovine RSV or murine pneumonia virus) toachieve a desired attenuation. As another example, a nucleotide sequenceat or near the cleavage site of the F protein, or the putativeattachment domain of the G protein, can be modified by point mutations,site-specific changes, or by alterations involving entire genes or genesegments to achieve novel effects on viral growth in tissue cultureand/or infection and pathogenesis in experimental animals.

Thus, infectious recombinant RSV intended for administration to humanscan be a human RSV that has been modified to contain genes from, e.g., abovine or murine RSV or a PIV, such as for the purpose of attenuation.For example, by inserting a gene or gene segment from PIV, a bivalentvaccine to both PIV and RSV is provided. Alternatively, a heterologousRSV species, subgroup or strain, or a distinct respiratory pathogen suchas PIV, may be modified, e.g., to contain genes that encode epitopes orproteins which elicit protection against human RSV infection. Forexample, the human RSV glycoprotein genes can be substituted for thebovine glycoprotein genes such that the resulting bovine RSV, which nowbears the human RSV surface glycoproteins and would retain a restrictedability to replicate in a human host due to the remaining bovine geneticbackground, elicits a protective immune response in humans against humanRSV strains.

The ability to analyze and incorporate other types of attenuatingmutations into infectious RSV for vaccine development extends to a broadassemblage of targeted changes in RSV clones. For example, deletion ofthe SH gene yields a recombinant RSV having novel phenotypiccharacteristics, including enhanced growth. In the present invention, anSH gene deletion (or any other selected, non-essential gene or genesegment deletion), is combined in a recombinant RSV with one or moreadditional mutations specifying an attenuated phenotype, e.g., a pointmutation adopted from a biologically derived attenuated RSV mutant. Inexemplary embodiments, the SH gene or NS2 gene is deleted in combinationwith one or more cp and/or ts mutations adopted from cpts248/404,cpts530/1009, cpts530/1030, or another selected mutant RSV strain, toyield a recombinant RSV having increased yield of virus, enhancedattenuation, and genetic to phenotypic reversion, due to the combinedeffects of the different mutations. In this regard, any RSV gene whichis not essential for growth, for example the SH, NP, NS1 and NS2 genes,can be ablated or otherwise modified to yield desired effects onvirulence, pathogenesis, immunogenicity and other phenotypic characters.For example, ablation by deletion of a non-essential gene such as SHresults in enhanced viral growth in culture. Without wishing to be boundby theory, this effect is likely due in part to a reduced nucleotidelength of the viral genome. In the case of one exemplary SH-minus clone,the modified viral genome is 14,825 nt long, 398 nucleotides less thanwild type. By engineering similar mutations that decrease genome size,e.g., in other coding or noncoding regions elsewhere in the RSV genome,such as in the P, M, F and M2 genes, the invention provides severalreadily obtainable methods and materials for improving RSV growth.

In addition, a variety of other genetic alterations can be produced in arecombinant RSV genome or antigenome for incorporation into infectiousrecombinant RSV, alone or together with one or more attenuating pointmutations adopted from a biologically derived mutant RSV. Heterologousgenes (e.g. from different RSV strains or types or non-RSV sources) maybe inserted in whole or in part, the order of genes changed, geneoverlap removed, the RSV genome promoter replaced with its antigenomecounterpart, portions of genes removed or substituted, and even entiregenes deleted. Different or additional modifications in the sequence canbe made to facilitate manipulations, such as the insertion of uniquerestriction sites in various intergenic regions (e.g., a unique Stulsite between the G and F genes) or elsewhere. Nontranslated genesequences can be removed to increase capacity for inserting foreignsequences.

In exemplary embodiments, individual genes, gene segments, or single ormultiple nucleotides of one RSV may be substituted by counterpartsequence(s) from a heterologous RSV or other source. For example, aselected, heterologous gene segment, such as one encoding a cytoplasmictail, transmembrane domain or ectodomain, an epitopic site or region, abinding site or region, an active site or region containing an activesite, etc., of a selected protein from one RSV, can be substituted for acounterpart gene segment in another RSV to yield novel recombinants, forexample recombinants expressing a chimeric protein having a cytoplasmictail and/or transmembrane domain of one RSV fused to an ectodomain ofanother RSV. Useful genome segments in this regard range from about15-35 nucleotides in the case of gene segments encoding small functionaldomains of proteins, e.g., epitopic sites, to about 50, 75, 100,200-500, and 500-1,500 or more nucleotides for gene segments encodinglarger domains or protein regions. In one embodiment, F and/or Gprotective antigens of one RSV strain or subgroup are substituted intoan RSV clone of a different strain or subgroup to produce a recombinantvirus capable of stimulating a cross-protective immune response againstboth strains or subgroups in an immunized host. In additional aspects, achimeric RSV clone having a mutation involving alteration of a gene orgene segment, e.g., a substituted, heterologous F and/or G gene or genesegment, is further modified by introducing one or more attenuating tsor cp point mutations adopted from a biologically derived mutant RSVstrain, e.g., cpts248/404, cpts530/1009, or cpts530/1030.

In yet additional aspects, one or more human RSV coding or non-codingpolynucleotides are substituted with a counterpart sequence from bovineor murine RSV, alone or in combination with one or more selected cp orts mutations, to yield novel attenuated vaccine strains. In oneembodiment, a chimeric bovine-human RSV incorporates a substitution ofthe human RSV NP gene or gene segment with a counterpart bovine NP geneor gene segment, which chimera can optionally be constructed toincorporate a SH, NP, NS1, NS2 or other gene deletion, one or more cp orts point mutations, or various combinations of these and other mutationsdisclosed herein. The replacement of a human RSV coding sequence (e.g.,of NS1, NS2, NP, etc.) or non-coding sequence (e.g., a promoter,gene-end, gene-start, intergenic or other cis-acting element) with acounterpart bovine or murine RSV sequence yields attenuated recombinantshaving a variety of possible attenuating effects. For example, a hostrange effect may arise from a heterologous RSV gene not functioningefficiently in a human cell, from incompatibility of the heterologoussequence or protein with a biologically interactive human RSV sequenceor protein (e.g., a sequence or protein that ordinarily cooperates withthe substituted sequence or protein for viral transcription,translation, assembly, etc.), among other useful attenuating effects.

In yet another aspect of the invention, insertion of foreign genes orgene segments, and in some cases of noncoding nucleotide sequences, intothe RSV genome results in a desired increase in genome length causingyet additional, desired phenotypic effects. Increased genome lengthresults in attenuation of the resultant RSV, dependent in part upon thelength of the insert. In addition, the expression of certain proteinsfrom a gene inserted into recombinant RSV will result in attenuation ofthe virus due to the action of the protein. This has been described forIL-2 expressed in vaccinia virus (e.g. Flexner et al., Nature 33:-259-62(1987)) and also would be expected for gamma interferon.

Deletions, insertions, substitutions and other mutations involvingchanges of whole viral genes or gene segments in recombinant RSV of theinvention yield highly stable vaccine candidates, which are particularlyimportant in the case of immunosuppressed individuals. Many of thesemutations will result in attenuation of resultant vaccine strains,whereas others will specify different types of desired phenotypicchanges. For example, certain viral genes are known which encodeproteins that specifically interfere with host immunity (see, e.g., Katoet al., EMBO. J. 16:578-87 (1997), incorporated herein by reference).Ablation of such genes in vaccine viruses is expected to reducevirulence and pathogenesis and/or improve immunogenicity.

Also provided within the invention are genetic modifications in arecombinant RSV which alter or ablate the expression of a selected geneor gene segment without removing the gene or gene segment from the RSVclone, e.g., by introducing a termination codon within a selected codingsequence; changing the position of a gene or introducing an upstreamstart codon to alter its rate of expression; changing GS and/or GEtranscription signals to alter phenotype (e.g., growth, temperaturerestrictions on transcription, etc.) Preferred mutations in this contextinclude mutations directed toward cis-acting signals, which can beidentified, e.g., by mutational analysis of RSV minigenomes. Forexample, insertional and deletional analysis of the leader and trailerand flanking sequences identified viral promoters and transcriptionsignals and provided a series of mutations associated with varyingdegrees of reduction of RNA replication or transcription. Saturationmutagenesis (whereby each position in turn is modified to each of thenucleotide alternatives) of these cis-acting signals also has identifiedmany mutations which reduced (or in one case increased) RNA replicationor transcription. Any of these mutations can be inserted into thecomplete antigenome or genome as described herein. Evaluation andmanipulation of trans-acting proteins and cis-acting RNA sequences usingthe complete antigenome cDNA is assisted by the use of RSV minigenomes(see, e.g., Grosfeld et al., J. Virol. 69: 5677-5686 (1995),incorporated herein by reference), whose helper-dependent status isuseful in the characterization of those mutants which are too inhibitoryto be recovered in replication-independent infectious virus.

Other mutations within RSV of the present invention involve replacementof the 3′ end of genome with its counterpart from antigenome, which isassociated with changes in RNA replication and transcription. Inaddition, the intergenic regions (Collins et al., Proc. Natl. Acad. Sci.USA 83:4594-4598 (1986), incorporated herein by reference) can beshortened or lengthened or changed in sequence content, and thenaturally-occurring gene overlap (Collins et al., Proc. Natl. Acad. Sci.USA 84:5134-5138 (1987), incorporated herein by reference) can beremoved or changed to a different intergenic region by the methodsdescribed herein.

In one exemplary embodiment, the level of expression of specific RSVproteins, such as the protective F and G antigens, can be increased bysubstituting the natural sequences with ones which have been madesynthetically and designed to be consistent with efficient translation.In this context, it has been shown that codon usage can be a majorfactor in the level of translation of mammalian viral proteins (Haas etal., Current Biol. 6:315-324 (1996)). Examination of the codon usage ofthe mRNAs encoding the F and G proteins of RSV, which are the majorprotective antigens, shows that the usage is consistent with poorexpression. Thus, codon usage can be improved by the recombinant methodsof the invention to achieve improved expression for selected genes.

In another exemplary embodiment, a sequence surrounding a translationalstart site (preferably including a nucleotide in the −3 position) of aselected RSV gene is modified, alone or in combination with introductionof an upstream start codon, to modulate RSV gene expression byspecifying up- or down-regulation of translation.

Alternatively, or in combination with other RSV modifications disclosedherein, RSV gene expression can be modulated by altering atranscriptional GS signal of a selected gene(s) of the virus. In oneexemplary embodiment, the GS signal of NS2 is modified to include adefined mutation (e.g., the 404(M2) mutation described hereinbelow) tosuperimpose a ts restriction on viral replication.

Yet additional RSV clones within the invention incorporate modificationsto a transcriptional GE signal. For example, RSV clones are providedwhich substitute or mutate the GE signal of the NS1 and NS2 genes forthat of the N gene, resulting in decreased levels of readthrough mRNAsand increased expression of proteins from downstream genes. Theresulting recombinant virus exhibits increased growth kinetics andincreased plaque size, providing but one example of alteration of RSVgrowth properties by modification of a cis-acting regulatory element inthe RSV genome.

In another exemplary embodiment, expression of the G protein isincreased by modification of the G mRNA. The G protein is expressed asboth a membrane bound and a secreted form, the latter form beingexpressed by translational initiation at a start site within the Gtranslational open reading frame. The secreted form can account for asmuch as one-half of the expressed G protein. Ablation of the internalstart site (e.g., by sequence alteration, deletion, etc.), alone ortogether with altering the sequence context of the upstream start siteyields desired changes in G protein expression. Ablation of the secretedform of G also will improve the quality of the host immune response toexemplary, recombinant RSV, because the soluble form of G is thought toact as a “decoy” to trap neutralizing antibodies. Also, soluble Gprotein has been implicated in enhanced immunopathology due to itspreferential stimulation of a Th2-biased response.

In alternative embodiments, levels of RSV gene expression are modifiedat the level of transcription. In one aspect, the position of a selectedgene in the RSV gene map can be changed to a more promoter-proximal orpromotor-distal position, whereby the gene will be expressed more orless efficiently, respectively. According to this aspect, modulation ofexpression for specific genes can be achieved yielding reductions orincreases of gene expression from two-fold, more typically four-fold, upto ten-fold or more compared to wild-type levels. In one example, theNS2 gene (second in order in the RSV gene map) is substituted inposition for the SH gene (sixth in order), yielding a predicted decreasein expression of NS2. Increased expression of selected RSV genes due topositional changes can be achieved up to 10-fold, 30-fold, 50-fold,100-fold or more, often attended by a commensurate decrease inexpression levels for reciprocally, positionally substituted genes.

In other exemplary embodiments, the F and G genes are transpositionedsingly or together to a more promoter-proximal or promoter-distal sitewithin the (recombinant) RSV gene map to achieve higher or lower levelsof gene expression, respectively. These and other transpositioningchanges yield novel RSV clones having attenuated phenotypes, for exampledue to decreased expression of selected viral proteins involved in RNAreplication.

In more detailed aspects of the invention, a recombinant RSV is providedin which expression of a viral gene, for example the NS2 gene, isablated at the translational level without deletion of the gene or of asegment thereof, by, e.g., introducing two tandem translationaltermination codons into a translational open reading frame (ORF). Thisyields viable virus in which a selected gene has been silenced at thelevel of translation, without deleting its gene. These forms of“knock-out” virus exhibit reduced growth rates and small plaque sizes intissue culture. Thus, the methods and compositions of the inventionprovide yet additional, novel types of RSV attenuating mutations whichablate expression of a viral gene that is not one of the major viralprotective antigens. In this context “knockout” virus phenotypesproduced without deletion of a gene or gene segment can be alternativelyproduced by deletion mutagenesis, as described herein, to effectivelypreclude correcting mutations that may restore synthesis of a targetprotein. Several other gene “knock-outs” for RSV can be made usingalternate designs. For example, insertion of translation terminationcodons into ORFs, or disruption of the RNA editing sites, offeralternatives to silencing or attenuating the expression of selectedgenes. Methods for producing these and other knock-outs are well knownin the art (as described, for example, in Kretzschmar et al., Virology216:309-316 (1996); Radecke et al., Virology 217:418-412 (1996); andKato et al., EMBO J. 16:178-587 (1987); and Schneider et al., Virology277:314-322 (1996), each incorporated herein by reference).

In yet other embodiments, RSV useful in a vaccine formulation can beconveniently modified to accommodate antigenic drift in circulatingvirus. Typically the modification will be in the G and/or F proteins.The entire G or F gene, or the segments encoding particular immunogenicregions thereof, is incorporated into the RSV genome or antigenome cDNAby replacement of the corresponding region in the infectious clone or byadding one or more copies of the gene such that several antigenic formsare represented. Progeny virus produced from the modified RSV cDNA arethen used in vaccination protocols against the emerging strains.Further, inclusion of the G protein gene of RSV subgroup B as a geneaddition will broaden the response to cover a wider spectrum of therelatively diverse subgroup A and B strains present in the humanpopulation.

An infectious RSV clone of the invention can also be engineeredaccording to the methods and compositions disclosed herein to enhanceits immunogenicity and induce a level of protection greater than thatprovided by infection with a wild-type RSV or an incompletely attenuatedparental virus or clone. For example, an immunogenic epitope from aheterologous RSV strain or type, or from a non-RSV source such as PIV,can be added by appropriate nucleotide changes in the polynucleotidesequence encoding the RSV genome or antigenome. Alternatively,recombinant RSV can be engineered to identify and ablate (e.g., by aminoacid insertion, substitution or deletion) epitopes associated withundesirable immunopathologic reactions. In other embodiments, anadditional gene is inserted into or proximate to the RSV genome orantigenome which is under the control of an independent set oftranscription signals. Genes of interest include those encodingcytokines (e.g., IL-2 through IL-15, especially IL-2, IL-6 and IL-12,etc.), gamma-interferon, and include those encoding cytokines (e.g.,IL-2 through IL-15, especially IL-2, IL-6 and IL-12, etc.),gamma-interferon, and proteins rich in T helper cell epitopes. Theadditional protein can be expressed either as a separate protein or as achimera engineered from a second copy of one of the RSV proteins, suchas SH. This provides the ability to modify and improve the immuneresponse against RSV both quantitatively and qualitatively.

In another aspect of the invention the recombinant RSV can be employedas a vector for protective antigens of other respiratory tractpathogens, such as parainfluenza virus (PIV), e.g., by incorporatingsequences encoding those protective antigens from PIV into the RSVgenome or antigenome used to produce infectious vaccine virus, asdescribed herein. The cloning of PIV cDNA and other disclosure isdescribed in United States Provisional Patent Application entitledPRODUCTION OF PARAINFLUENZA VIRUS FROM CLONED NUCLEOTIDE SEQUENCES,filed May 23, 1997, Ser. No. 60/047,575 and incorporated herein byreference. In alternate embodiments, a modified RSV is provided whichcomprises a chimera of a RSV genomic or antigenomic sequence and atleast one PIV sequence, for example a polynucleotide containingsequences from both RSV and PIV1, PIV2, PIV3 or bovine PIV. For example,individual genes of RSV may be replaced with counterpart genes fromhuman PIV, such as the HN and/or F glycoprotein genes of PIV1, PIV2, orPIV3. Alternatively, a selected, heterologous gene segment, such as acytoplasmic tail, transmembrane domain or ectodomain of HN or F ofHPIV1, HPIV2, or HPIV3 can be substituted for a counterpart gene segmentin, e.g., the same gene in an RSV clone, within a different gene in theRSV clone, or into a non-coding sequence of the RSV genome orantigenome. In one embodiment, a gene segment from HN or F of HPIV3 issubstituted for a counterpart gene segment in RSV type A, to yieldconstructs encoding chimeric proteins, e.g. fusion proteins having acytoplasmic tail and/or transmembrane domain of RSV fused to anectodomain of RSV to yield a novel attenuated virus, and/or amultivalent vaccine immunogenic against both PIV and RSV.

In addition to the above described modifications to recombinant RSV,different or additional modifications in RSV clones can be made tofacilitate manipulations, such as the insertion of unique restrictionsites in various intergenic regions (e.g., a unique Stul site betweenthe G and F genes) or elsewhere. Nontranslated gene sequences can beremoved to increase capacity for inserting foreign sequences.

Introduction of the foregoing, defined mutations into an infectious RSVclone can be achieved by a variety of well known methods. By “infectiousclone” is meant cDNA or its product, synthetic or otherwise, which canbe transcribed into genomic or antigenomic RNA capable of serving astemplate to produce the genome of an infectious virus or subviralparticle. Thus, defined mutations can be introduced by conventionaltechniques (e.g., site-directed mutagenesis) into a cDNA copy of thegenome or antigenome. The use of antigenome or genome cDNA subfragmentsto assemble a complete antigenome or genome cDNA as described herein hasthe advantage that each region can be manipulated separately (smallercDNAs are easier to manipulate than large ones) and then readilyassembled into a complete cDNA. Thus, the complete antigenome or genomecDNA, or any subfragment thereof, can be used as template foroligonucleotide-directed mutagenesis. This can be through theintermediate of a single-stranded phagemid form, such as using theMuta-gene® kit of Bio-Rad Laboratories (Richmond, Calif.) or a methodusing the double-stranded plasmid directly as template such as theChameleon mutagenesis kit of Stratagene (La Jolla, Calif.) or by thepolymerase chain reaction employing either an oligonucleotide primer ortemplate which contains the mutation(s) of interest. A mutatedsubfragment can then be assembled into the complete antigenome or genomecDNA. A variety of other mutagenesis techniques are known and availablefor use in producing the mutations of interest in the RSV antigenome orgenome cDNA. Mutations can vary from single nucleotide changes toreplacement of large cDNA pieces containing one or more genes or genomeregions.

Thus, in one illustrative embodiment mutations are introduced by usingthe Muta-gene phagemid in vitro mutagenesis kit available from Bio-Rad.In brief, cDNA encoding a portion of an RSV genome or antigenome iscloned into the plasmid pTZ18U, and used to transform CJ236 cells (LifeTechnologies). Phagemid preparations are prepared as recommended by themanufacturer. Oligonucleotides are designed for mutagenesis byintroduction of an altered nucleotide at the desired position of thegenome or antigenome. The plasmid containing the genetically alteredgenome or antigenome fragment is then amplified and the mutated piece isthen reintroduced into the full-length genome or antigenome clone.

The ability to introduce defined mutations into infectious RSV has manyapplications, including the analyses of RSV molecular biology andpathogenesis. For example, the functions of the RSV proteins, includingthe NS1, NS2, SH, M2(ORF1) and M2(ORF2) proteins, can be investigatedand manipulated by introducing mutations which ablate or reduce theirlevel of expression, or which yield mutant protein. In one exemplaryembodiment hereinbelow, RSV virus was constructed in which expression ofa viral gene, namely the SH gene, was ablated by deletion of the mRNAcoding sequence and flanking transcription signals. Surprisingly, notonly could this virus be recovered, but it grew efficiently in tissueculture. In fact, its growth was substantially increased over that ofthe wild type, based on both yield of infectious virus and on plaquesize. This improved growth in tissue culture from the SH deletion andother RSV derivatives of the invention provides useful tools fordeveloping RSV vaccines, which overcome the problem of RSV's poor yieldin tissue culture that had complicated production of vaccine virus inother systems. These deletions are highly stable against geneticreversion, rendering the RSV clones derived therefrom particularlyuseful as vaccine agents.

The invention also provides methods for producing an infectious RSV fromone or more isolated polynucleotides, e.g., one or more cDNAs. Accordingto the present invention cDNA encoding a RSV genome or antigenome isconstructed for intracellular or in vitro coexpression with thenecessary viral proteins to form infectious RSV. By “RSV antigenome” ismeant an isolated positive-sense polynucleotide molecule which serves asthe template for the synthesis of progeny RSV genome. Preferably a cDNAis constructed which is a positive-sense version of the RSV genome,corresponding to the replicative intermediate RNA, or antigenome, so asto minimize the possibility of hybridizing with positive-sensetranscripts of the complementing sequences that encode proteinsnecessary to generate a transcribing, replicating nucleocapsid, i.e.,sequences that encode N, P, L and M2(ORF1) protein. In an RSV minigenomesystem, genome and antigenome were equally active in rescue, whethercomplemented by RSV or by plasmids, indicating that either genome orantigenome can be used and thus the choice can be made on methodologicor other grounds.

A native RSV genome typically comprises a negative-sense polynucleotidemolecule which, through complementary viral mRNAs, encodes elevenspecies of viral proteins, i.e., the nonstructural species NS1 and NS2,N, P, matrix (M), small hydrophobic (SH), glycoprotein (G), fusion (F),M2(ORF1), M2(ORF2), and L, substantially as described in Mink et al.,Virology 185: 615-624 (1991), Stec et al., Virology 183: 273-287 (1991),and Connors et al., Virol. 208:478-484 (1995), incorporated herein byreference. For purposes of the present invention the genome orantigenome of the recombinant RSV of the invention need only containthose genes or portions thereof necessary to render the viral orsubviral particles encoded thereby infectious. Further, the genes orportions thereof may be provided by more than one polynucleotidemolecule, i.e., a gene may be provided by complementation or the likefrom a separate nucleotide molecule.

By recombinant RSV is meant a RSV or RSV-like viral or subviral particlederived directly or indirectly from a recombinant expression system orpropagated from virus or subviral particles produced therefrom. Therecombinant expression system will employ a recombinant expressionvector which comprises an operably linked transcriptional unitcomprising an assembly of at least a genetic element or elements havinga regulatory role in RSV gene expression, for example, a promoter, astructural or coding sequence which is transcribed into RSV RNA, andappropriate transcription initiation and termination sequences.

To produce infectious RSV from cDNA-expressed genome or antigenome, thegenome or antigenome is coexpressed with those RSV proteins necessary to(i) produce a nucleocapsid capable of RNA replication, and (ii) renderprogeny nucleocapsids competent for both RNA replication andtranscription. Transcription by the genome nucleocapsid provides theother RSV proteins and initiates a productive infection. Alternatively,additional RSV proteins needed for a productive infection can besupplied by coexpression.

An RSV antigenome may be constructed for use in the present inventionby, e.g., assembling cloned cDNA segments, representing in aggregate thecomplete antigenome, by polymerase chain reaction (PCR; described in,e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202, and PCR Protocols: A Guideto Methods and Applications, Innis et al., eds., Academic Press, SanDiego (1990), incorporated herein by reference) of reverse-transcribedcopies of RSV mRNA or genome RNA. For example, cDNAs containing thelefthand end of the antigenome, spanning from an appropriate promoter(e.g., T7 RNA polymerase promoter) and the leader region complement tothe SH gene, are assembled in an appropriate expression vector, such asa plasmid (e.g., pBR322) or various available cosmid, phage, or DNAvirus vectors. The vector may be modified by mutagenesis and/orinsertion of synthetic polylinker containing unique restriction sitesdesigned to facilitate assembly. For example, a plasmid vector describedherein was derived from pBR322 by replacement of the PstI-EcoR1 fragmentwith a synthetic DNA containing convenient restriction enzyme sites. Useof pBR322 as a vector stabilized nucleotides 3716-3732 of the RSVsequence, which otherwise sustained nucleotide deletions or insertions,and propagation of the plasmid was in bacterial strain DH10B to avoid anartifactual duplication and insertion which otherwise occurred in thevicinity of nt 4499. For ease of preparation the G, F and M2 genes canbe assembled in a separate vector, as can be the L and trailersequences. The righthand end (e.g., L and trailer sequences) of theantigenome plasmid may contain additional sequences as desired, such asa flanking ribozyme and tandem T7 transcriptional terminators. Theribozyme can be hammerhead type (e.g., Grosfeld et al., J. Virol.69:5677-5686 (1995)), which would yield a 3′ end containing a singlenonviral nucleotide, or can any of the other suitable ribozymes such asthat of hepatitis delta virus (Perrotta et al., Nature 350:434-436(1991)) which would yield a 3′ end free of non-RSV nucleotides. A middlesegment (e.g., G-to-M2 piece) is inserted into an appropriaterestriction site of the leader-to-SH plasmid, which in turn is therecipient for the L-trailer-ribozyme-terminator piece, yielding acomplete antigenome. In an illustrative example described herein, theleader end was constructed to abut the promoter for T7 RNA polymerasewhich included three transcribed G residues for optimal activity;transcription donates these three nonviral G's to the 5′ end of theantigenome. These three nonviral G residues can be omitted to yield a 5′end free of nonviral nucleotides. To generate a nearly-correct 3′ end,the trailer end was constructed to be adjacent to a hammerhead ribozyme,which upon cleavage would donate a single 3′-phosphorylated U residue tothe 3′ end of the encoded RNA.

In certain embodiments of the invention, complementing sequencesencoding proteins necessary to generate a transcribing, replicating RSVnucleocapsid are provided by one or more helper viruses. Such helperviruses can be wild type or mutant. Preferably, the helper virus can bedistinguished phenotypically from the virus encoded by the RSV cDNA. Forexample, it is desirable to provide monoclonal antibodies which reactimmunologically with the helper virus but not the virus encoded by theRSV cDNA. Such antibodies can be neutralizing antibodies. In someembodiments, the antibodies can be used in affinity chromatography toseparate the helper virus from the recombinant virus. To aid theprocurement of such antibodies, mutations can be introduced into the RSVcDNA to provide antigenic diversity from the helper virus, such as inthe HN or F glycoprotein genes.

A variety of nucleotide insertions and deletions can be made in the RSVgenome or antigenome. The nucleotide length of the genome of wild typehuman RSV (15,222 nucleotides) is a multiple of six, and members of theParamyxovirus and Morbillivirus genera typically abide by a “rule ofsix,” i.e., genomes (or minigenomes) replicate efficiently only whentheir nucleotide length is a multiple of six (thought to be arequirement for precise spacing of nucleotide residues relative toencapsidating NP protein). Alteration of RSV genome length by singleresidue increments had no effect on the efficiency of replication, andsequence analysis of several different minigenome mutants followingpassage showed that the length differences were maintained withoutcompensatory changes. Thus, RSV lacks the strict requirement of genomelength being a multiple of six, and nucleotide insertions and deletionscan be made in the RSV genome or antigenome without defeatingreplication of the recombinant RSV of the present invention.

Alternative means to construct cDNA encoding the genome or antigenomeinclude by reverse transcription-PCR using improved PCR conditions(e.g., as described in Cheng et al., Proc. Natl. Acad. Sci. USA91:5695-5699 (1994); Samal et al., J. Virol 70:5075-5082 (1996), eachincorporated herein by reference) to reduce the number of subunit cDNAcomponents to as few as one or two pieces. In other embodimentsdifferent promoters can be used (e.g., T3, SP6) or different ribozymes(e.g., that of hepatitis delta virus. Different DNA vectors (e.g.,cosmids) can be used for propagation to better accommodate the largesize genome or antigenome.

The N, P and L proteins, necessary for RNA replication, require an RNApolymerase elongation factor such as the M2(ORF1) protein for processivetranscription. Thus M2(ORF1) or a substantially equivalent transcriptionelongation factor for negative strand RNA viruses is required for theproduction of infectious RSV and is a necessary component of functionalnucleocapsids during productive infection. The need for the M2(ORF1)protein is consistent with its role as a transcription elongationfactor. The need for expression of the RNA polymerase elongation factorprotein for negative strand RNA viruses is a feature of the presentinvention. M2(ORF1) can be supplied by expression of the completeM2-gene, although in this form the second ORF2 may also be expressed andhave an inhibitory effect on RNA replication. Therefore, for productionof infectious virus using the complete M2 gene the activities of the twoORFs should be balanced to permit sufficient expression of M(ORF1) toprovide transcription elongation activity yet not so much of M(ORF2) toinhibit RNA replication. Alternatively, the ORF1 protein is providedfrom a cDNA engineered to lack ORF2 or which encodes a defective ORF2.Efficiency of virus production may also be improved by co-expression ofadditional viral protein genes, such as those encoding envelopeconstituents (i.e., SH, M, G, F proteins).

Isolated polynucleotides (e.g., cDNA) encoding the genome or antigenomeand, separately, the N, P, L and M2(ORF1) proteins, are inserted bytransfection, electroporation, mechanical insertion, transduction or thelike, into cells which are capable of supporting a productive RSVinfection, e.g., HEp-2, FRhL-DBS2, MRC, and Vero cells. Transfection ofisolated polynucleotide sequences may be introduced into cultured cellsby, for example, calcium phosphate-mediated transfection (Wigler et al.,Cell 14: 725 (1978); Corsaro and Pearson, Somatic Cell Genetics 7: 603(1981); Graham and Van der Eb, Virology 52: 456 (1973)), electroporation(Neumann et al., EMBO J. 1: 841-845 (1982)), DEAE-dextran mediatedtransfection (Ausubel et al., (ed.) Current Protocols in MolecularBiology, John Wiley and Sons, Inc., NY (1987), cationic lipid-mediatedtransfection (Hawley-Nelson et al., Focus 15:73-79 (1993)) or acommercially available transfection regent, e.g., LipofectACE® (LifeTechnologies) (each of the foregoing references are incorporated hereinby reference).

The N, P, L and M2(ORF1) proteins are encoded by one or more expressionvectors which can be the same or separate from that which encodes thegenome or antigenome, and various combinations thereof. Additionalproteins may be included as desired, encoded by its own vector or by avector encoding a N, P, L, or M2(ORF1) protein or the complete genome orantigenome. Expression of the genome or antigenome and proteins fromtransfected plasmids can be achieved, for example, by each cDNA beingunder the control of a promoter for T7 RNA polymerase, which in turn issupplied by infection, transfection or transduction with an expressionsystem for the T7 RNA polymerase, e.g., a vaccinia virus MVA strainrecombinant which expresses the T7 RNA polymerase (Wyatt et al.,Virology, 210:202-205 (1995), incorporated herein by reference). Theviral proteins, and/or T7 RNA polymerase, can also be provided fromtransformed mammalian cells, or by transfection of preformed mRNA orprotein.

Alternatively, synthesis of antigenome or genome can be done in vitro(cell-free) in a combined transcription-translation reaction, followedby transfection into cells. Or, antigenome or genome RNA can besynthesized in vitro and transfected into cells expressing RSV proteins.

To select candidate vaccine viruses from the host of biologicallyderived and recombinant RSV strains provided herein, the criteria ofviability, attenuation and immunogenicity are determined according towell known methods. Viruses which will be most desired in vaccines ofthe invention must maintain viability, have a stable attenuationphenotype, exhibit replication in an immunized host (albeit at lowerlevels), and effectively elicit production of an immune response in avaccinee sufficient to confer protection against serious disease causedby subsequent infection from wild-type virus. Clearly, the heretoforeknown and reported RS virus mutants do not meet all of these criteria.Indeed, contrary to expectations based on the results reported for knownattenuated RSV, viruses of the invention are not only viable and moreattenuated then previous mutants, but are more stable genetically invivo than those previously studied mutants, retaining the ability tostimulate a protective immune response and in some instances to expandthe protection afforded by multiple modifications, e.g., induceprotection against different viral strains or subgroups, or protectionby a different immunologic basis, e.g., secretory versus serumimmunoglobulins, cellular immunity, and the like. Prior to theinvention, genetic instability of the ts phenotype following replicationin vivo has been the rule for ts viruses (Murphy et al., Infect. Immun.37:235-242 (1982)).

To propagate a RSV virus for vaccine use and other purposes, a number ofcell lines which allow for RSV growth may be used. RSV grows in avariety of human and animal cells. Preferred cell lines for propagatingattenuated RS virus for vaccine use include DBS-FRhL-2, MRC-5, and Verocells. Highest virus yields are usually achieved with epithelial celllines such as Vero cells. Cells are typically inoculated with virus at amultiplicity of infection ranging from about 0.001 to 1.0, or more, andare cultivated under conditions permissive for replication of the virus,e.g., at about 30-37° C. and for about 3-5 days, or as long as necessaryfor virus to reach an adequate titer. Virus is removed from cell cultureand separated from cellular components, typically by well knownclarification procedures, e.g., centrifugation, and may be furtherpurified as desired using procedures well known to those skilled in theart.

RSV which has been attenuated as described herein can be tested invarious well known and generally accepted in vitro and in vivo models toconfirm adequate attenuation, resistance to phenotypic reversion, andimmunogenicity for vaccine use. In in vitro assays, the modified virus(e.g., a multiply attenuated, biologically derived or recombinant RSV)is tested for temperature sensitivity of virus replication, i.e. tsphenotype, and for the small plaque phenotype. Modified viruses arefurther tested in animal models of RSV infection. A variety of animalmodels have been described and are summarized in Meignier et al., eds.,Animal Models of Respiratory Syncytial Virus Infection, MerieuxFoundation Publication, (1991), which is incorporated herein byreference. A cotton rat model of RS infection is described in U.S. Pat.No. 4,800,078 and Prince et al., Virus Res. 3:193-206 (1985), which areincorporated herein by reference, and is believed to be predictive ofattenuation and efficacy in humans. A primate model of RSV infectionusing the chimpanzee is predictive of attenuation and efficacy inhumans, and is described in detail in Richardson et al., J. Med. Virol.3:91-100 (1978); Wright et al., Infect. Immun., 37:397-400 (1982); Croweet al., Vaccine 11:1395-1404 (1993), which are incorporated herein byreference.

The interrelatedness of data derived from rodents and chimpanzeesrelating to the level of attenuation of RSV candidates can bedemonstrated by reference to FIG. 1, which is a graph correlating thereplication of a spectrum of respiratory syncytial subgroup A viruses inthe lungs of mice with their replication in chimpanzees. The relativelevel of replication compared to that of wt RSV is substantiallyidentical, allowing the mouse to serve as a model in which to initiallycharacterize the level of attenuation of the vaccine RSV candidate. Themouse and cotton rat models are especially useful in those instances inwhich candidate RS viruses display inadequate growth in chimpanzees. TheRSV subgroup B viruses are an example of the RS viruses which growpoorly in chimpanzees.

Moreover, the therapeutic effect of RSV neutralizing antibodies ininfected cotton rats has been shown to be highly relevant to subsequentexperience with immunotherapy of monkeys and humans infected with RSV.Indeed, the cotton rat appears to be a reliable experimental surrogatefor the response of infected monkeys, chimpanzees and humans toimmunotherapy with RSV neutralizing antibodies. For example, the amountof RSV neutralizing antibodies associated with a therapeutic effect incotton rats as measured by the level of such antibodies in the serum oftreated animals (i.e., serum RSV neutralization titer of 1:302 to 1:518)is in the same range as that demonstrated for monkeys (i.e., titer of1:539) or human infants or small children (i.e., 1:877). A therapeuticeffect in cotton rats was manifest by a one hundred fold or greaterreduction in virus titer in the lung (Prince et al., J. Virol.61:1851-1854) while in monkeys a therapeutic effect was observed to be a50-fold reduction in pulmonary virus titer. (Hemming et al., J. Infect.Dis. 152:1083-1087 (1985)). Finally, a therapeutic effect in infants andyoung children who were hospitalized for serious RSV bronchiolitis orpneumonia was manifest by a significant increase in oxygenation in thetreated group and a significant decrease in amount of RSV recoverablefrom the upper respiratory tract of treated patients. (Hemming et al.,Antimicrob. Agents Chemother. 31:1882-1886 (1987)). Therefore, based onthese studies, the cotton rat constitutes a relevant model forpredicting success of RSV vaccines in infants and small children. Otherrodents, including mice, will also be similarly useful because theseanimals are permissive for RSV replication and have a core temperaturemore like that of humans (Wright et al., J. Infect. Dis. 122:501-512(1970) and Anderson et al., J. Gen. Virol. 71:(1990)).

In accordance with the foregoing description and based on the Examplesbelow, the invention also provides isolated, infectious RSV compositionsfor vaccine use. The attenuated virus which is a component of a vaccineis in an isolated and typically purified form. By isolated is meant torefer to RSV which is in other than a native environment of a wild-typevirus, such as the nasopharynx of an infected individual. Moregenerally, isolated is meant to include the attenuated virus as acomponent of a cell culture or other artificial medium. For example,attenuated RSV of the invention may be produced by an infected cellculture, separated from the cell culture and added to a stabilizer whichcontains other non-naturally occurring RS viruses, such as those whichare selected to be attenuated by means of resistance to neutralizingmonoclonal antibodies to the F-protein.

RSV vaccines of the invention contain as an active ingredient animmunogenically effective amount of RSV produced as described herein.Biologically derived or recombinant RSV can be used directly in vaccineformulations, or lyophilized. Lyophilized virus will typically bemaintained at about 4° C. When ready for use the lyophilized virus isreconstituted in a stabilizing solution, e.g., saline or comprising SPG,Mg⁺⁺ and HEPES, with or without adjuvant, as further described below.The biologically derived or recombinantly modified virus may beintroduced into a host with a physiologically acceptable carrier and/oradjuvant. Useful carriers are well known in the art, and include, e.g.,water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid andthe like. The resulting aqueous solutions may be packaged for use as is,or lyophilized, the lyophilized preparation being combined with asterile solution prior to administration, as mentioned above. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents, wettingagents and the like, for example, sodium acetate, sodium lactate, sodiumchloride, potassium chloride, calcium chloride, sorbitan monolaurate,triethanolamine oleate, and the like. Acceptable adjuvants includeincomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, oralum, which are materials well known in the art. Preferred adjuvantsalso include Stimulon™ QS-21 (Aquila Biopharmaceuticals, Inc.,Worchester, Mass.), MPL™ (3-0-deacylated monophosphoryl lipid A; RIBIImmunoChem Research, Inc., Hamilton, Mont.), and interleukin-12(Genetics Institute, Cambridge, Mass.).

Upon immunization with a RSV vaccine composition as described herein,via aerosol, droplet, oral, topical or other route, the immune system ofthe host responds to the vaccine by producing antibodies specific forRSV virus proteins, e.g., F and G glycoproteins. As a result of thevaccination the host becomes at least partially or completely immune toRSV infection, or resistant to developing moderate or severe RSVdisease, particularly of the lower respiratory tract.

The host to which the vaccine is administered can be any mammalsusceptible to infection by RSV or a closely related virus and capableof generating a protective immune response to antigens of thevaccinizing strain. Thus, suitable hosts include humans, non-humanprimates, bovine, equine, swine, ovine, caprine, lagamorph, rodents,etc. Accordingly, the invention provides methods for creating vaccinesfor a variety of human and veterinary uses.

The vaccine compositions containing the attenuated RSV of the inventionare administered to a patient susceptible to or otherwise at risk of RSvirus infection in an “immunogenically effective dose” which issufficient to induce or enhance the individual's immune responsecapabilities against RSV. In the case of human subjects, the attenuatedvirus of the invention is administered according to well establishedhuman RSV vaccine protocols, as described in, e.g., Wright et al.,Infect Immun. 37:397-400 (1982), Kim et al., Pediatrics 52:56-63 (1973),and Wright et al., J. Pediatr. 88:931-936 (1976), which are eachincorporated herein by reference. Briefly, adults or children areinoculated intranasally via droplet with an immunogenically effectivedose of RSV vaccine, typically in a volume of 0.5 ml of aphysiologically acceptable diluent or carrier. This has the advantage ofsimplicity and safety compared to parenteral immunization with anon-replicating vaccine. It also provides direct stimulation of localrespiratory tract immunity, which plays a major role in resistance toRSV. Further, this mode of vaccination effectively bypasses theimmunosuppressive effects of RSV-specific maternally-derived serumantibodies, which typically are found in the very young. Also, while theparenteral administration of RSV antigens can sometimes be associatedwith immunopathologic complications, this has never been observed with alive virus.

In all subjects, the precise amount of RSV vaccine administered and thetiming and repetition of administration will be determined based on thepatient's state of health and weight, the mode of administration, thenature of the formulation, etc. Dosages will generally range from about10³ to about 10⁶ plaque forming units (PFU) or more of virus perpatient, more commonly from about 10⁴ to 10⁵ PFU virus per patient. Inany event, the vaccine formulations should provide a quantity ofattenuated RSV of the invention sufficient to effectively stimulate orinduce an anti-RSV immune response, e.g., as can be determined bycomplement fixation, plaque neutralization, and/or enzyme-linkedimmunosorbent assay, among other methods. In this regard, individualsare also monitored for signs and symptoms of upper respiratory illness.As with administration to chimpanzees, the attenuated virus of thevaccine grows in the nasopharynx of vaccinees at levels approximately10-fold or more lower than wild-type virus, or approximately 10-fold ormore lower when compared to levels of incompletely attenuated RSV.

In neonates and infants, multiple administration may be required toelicit sufficient levels of immunity. Administration should begin withinthe first month of life, and at intervals throughout childhood, such asat two months, six months, one year and two years, as necessary tomaintain sufficient levels of protection against native (wild-type) RSVinfection. Similarly, adults who are particularly susceptible torepeated or serious RSV infection, such as, for example, health careworkers, day care workers, family members of young children, theelderly, individuals with compromised cardiopulmonary function, mayrequire multiple immunizations to establish and/or maintain protectiveimmune responses. Levels of induced immunity can be monitored bymeasuring amounts of neutralizing secretory and serum antibodies, anddosages adjusted or vaccinations repeated as necessary to maintaindesired levels of protection. Further, different vaccine viruses may beindicated for administration to different recipient groups. For example,an engineered RSV strain expressing a cytokine or an additional proteinrich in T cell epitopes may be particularly advantageous for adultsrather than for infants. RSV vaccines produced in accordance with thepresent invention can be combined with viruses of the other subgroup orstrains of RSV to achieve protection against multiple RSV subgroups orstrains, or selected gene segments encoding, e.g., protective epitopesof these strains can be engineered into one RSV clone as describedherein. Typically the different viruses will be in admixture andadministered simultaneously, but may also be administered separately.For example, as the F glycoproteins of the two RSV subgroups differ byonly about 11% in amino acid sequence, this similarity is the basis fora cross-protective immune response as observed in animals immunized withRSV or F antigen and challenged with a heterologous strain. Thus,immunization with one strain may protect against different strains ofthe same or different subgroup.

The vaccines of the invention elicit production of an immune responsethat is protective against serious lower respiratory tract disease, suchas pneumonia and bronchiolitis when the individual is subsequentlyinfected with wild-type RSV. While the naturally circulating virus isstill capable of causing infection, particularly in the upperrespiratory tract, there is a very greatly reduced possibility ofrhinitis as a result of the vaccination and possible boosting ofresistance by subsequent infection by wild-type virus. Followingvaccination, there are detectable levels of host engendered serum andsecretory antibodies which are capable of neutralizing homologous (ofthe same subgroup) wild-type virus in vitro and in vivo. In manyinstances the host antibodies will also neutralize wild-type virus of adifferent, non-vaccine subgroup.

The attenuated RS virus of the present invention exhibits a verysubstantial diminution of virulence when compared to wild-type virusthat is circulating naturally in humans. The attenuated virus issufficiently attenuated so that symptoms of infection will not occur inmost immunized individuals. In some instances the attenuated virus maystill be capable of dissemination to unvaccinated individuals. However,its virulence is sufficiently abrogated such that severe lowerrespiratory tract infections in the vaccinated or incidental host do notoccur.

The level of attenuation of vaccine virus may be determined by, forexample, quantifying the amount of virus present in the respiratorytract of an immunized host and comparing the amount to that produced bywild-type RSV or other attenuated RS viruses which have been evaluatedas candidate vaccine strains. For example, the attenuated virus of theinvention will have a greater degree of restriction of replication inthe upper respiratory tract of a highly susceptible host, such as achimpanzee, compared to the levels of replication of wild-type virus,e.g., 10- to 1000-fold less. Also, the level of replication of theattenuated RSV vaccine strain in the upper respiratory tract of thechimpanzee should be less than that of the RSV A2 ts-1 mutant, which wasdemonstrated previously to be incompletely attenuated in seronegativehuman infants. In order to further reduce the development of rhinorrhea,which is associated with the replication of virus in the upperrespiratory tract, an ideal vaccine candidate virus should exhibit arestricted level of replication in both the upper and lower respiratorytract. However, the attenuated viruses of the invention must besufficiently infectious and immunogenic in humans to confer protectionin vaccinated individuals. Methods for determining levels of RS virus inthe nasopharynx of an infected host are well known in the literature.Specimens are obtained by aspiration or washing out of nasopharyngealsecretions and virus quantified in tissue culture or other by laboratoryprocedure. See, for example, Belshe et al., J. Med. Virology 1:157-162(1977), Friedewald et al., J. Amer. Med. Assoc. 204:690-694 (1968);Gharpure et al., J. Virol. 3:414-421 (1969); and Wright et al., Arch.Ges. Virusforsch. 41:238-247 (1973). The virus can conveniently bemeasured in the nasopharynx of host animals, such as chimpanzees.

In some instances it may be desirable to combine the RSV vaccines of theinvention with vaccines which induce protective responses to otheragents, particularly other childhood viruses. For example, the RSVvaccine of the present invention can be administered simultaneously withparainfluenza virus vaccine, such as described in Clements et al., J.Clin. Microbiol. 29:1175-1182 (1991), which is incorporated herein byreference. In another aspect of the invention the RSV can be employed asa vector for protective antigens of other respiratory tract pathogens,such as parainfluenza, by incorporating the sequences encoding thoseprotective antigens into the RSV genome or antigenome which is used toproduce infectious RSV, as described herein.

In yet another aspect of the invention recombinant RSV is employed as avector for transient gene therapy of the respiratory tract. According tothis embodiment the recombinant RSV genome or antigenome incorporates asequence which is capable of encoding a gene product of interest. Thegene product of interest is under control of the same or a differentpromoter from that which controls RSV expression. The infectious RSVproduced by coexpressing the recombinant RSV genome or antigenome withthe N, P, L and M2(ORF1) proteins and containing a sequence encoding thegene product of interest is administered to a patient. Administration istypically by aerosol, nebulizer, or other topical application to therespiratory tract of the patient being treated. Recombinant RSV isadministered in an amount sufficient to result in the expression oftherapeutic or prophylactic levels of the desired gene product. Examplesof representative gene products which are administered in this methodinclude those which encode, for example, those particularly suitable fortransient expression, e.g., interleukin-2, interleukin-4,gamma-interferon, GM-CSF, G-CSF, erythropoietin, and other cytokines,glucocerebrosidase, phenylalanine hydroxylase, cystic fibrosistransmembrane conductance regulator (CFTR), hypoxanthine-guaninephosphoribosyl transferase, cytotoxins, tumor suppressor genes,antisense RNAs, and vaccine antigens.

The following examples are provided by way of illustration, notlimitation.

EXAMPLE I Isolation and Characterization of Mutagenized Derivatives ofCold-Passaged RSV

This Example describes the chemical mutagenesis of incompletelyattenuated host range-restricted cpRSV to produce derivative ts and spmutations which are more highly attenuated and thus are preferred foruse in RSV vaccine preparations.

A parent stock of cold-passaged RSV (cpRSV) was prepared. FlowLaboratories Lot 3131 virus, the cpRSV parent virus that is incompletelyattenuated in humans, was passaged twice in MRC-5 cells at 25° C.,terminally diluted twice in MRC-5 cells at 25° C., then passaged threetimes in MRC-5 to create cpRSV suspension for mutagenesis.

The cpRSV was mutagenized by growing the parent stock in MRC-5 cells at32° C. in the presence of 5-fluorouracil in the medium at aconcentration of 4×10⁻⁴M. This concentration was demonstrated to beoptimal in preliminary studies, as evidenced by a 100-fold decrease invirus titer on day 5 of growth in cell culture, compared to mediumwithout 5-fluorouracil. The mutagenized stock was then analyzed byplaque assay on Vero cells that were maintained under an agar overlay,and after an appropriate interval of incubation, plaques were stainedwith neutral red dye. 854 plaques were picked and the progeny of eachplaque were separately amplified by growth on fresh monolayers of Verocells. The contents of each of the tissue cultures inoculated with theprogeny of a single plaque of cpRSV-mutagenized virus were separatelyharvested when cytopathic effects on the Vero cells appeared maximal.Progeny virus that exhibited the temperature-sensitive (ts) orsmall-plaque (sp) phenotype was sought by titering these plaque pools onHEp-2 cells at 32° C. and 38° C. Any virus exhibiting a sp phenotype(plaque size that was reduced by 50% or more compared to parental virusat 32° C.) or a ts phenotype (100-fold or greater reduction in titer atrestrictive temperature [37° to 40° C.] compared to 32° C.) wasevaluated further. These strains were biologically cloned by serialplaque-purification on Vero cells three times, then amplified on Verocells. The cloned strains were titered at 32°, 37°, 38°, 39° and 40° C.(in an efficiency of plaque formation (EOP) assay) to confirm their spand ts phenotypes. Because titers of some cloned strains were relativelylow even at the permissive temperature (32°), these viruses werepassaged once in HEp-2 cells to create virus suspensions for in vitroanalysis. The phenotypes of the progeny of the mutagenized cpRSV arepresented on Table 1. TABLE 1 The efficiency of plaque formation of ninederivatives of cold- passaged RSV (cpts or cpsp mutants) in HEp-2 cellsat permissive and restrictive temperatures Shut-off The titer of virus(log₁₀pfu/ml) temper- Small- at the indicated temperature (° C.) atureplaques Virus 32 37 38 39 40 (° C.)¹ at 32 C. A2 4.5 4.4 4.5 3.8 3.8 >40no wild-type cp-RSV 6.0 5.8 5.8 6.2 5.4 >40 no ts-1 5.7 4.5 2.7 2.4 1.7*38 no cpsp143 4.2* 4.1* 3.8* 3.9* 3.8* >40 yes cpts368 6.7 6.3 6.1*5.8** 2.0** 40 no cpts274 7.3 7.1 6.6 5.8* 1.0** 40 no cpts347 6.2 6.15.7* 5.5** <0.7 40 no cpts142 5.7 5.1 4.5* 3.7** <0.7 39 no cpts299 6.25.5 5.1* 2.0** <0.7 39 no cpts475 5.4 4.8* 4.2** <0.7 <0.7 39 no cpts5305.5 4.8* 4.5* <0.7 <0.7 39 no cpts248 6.3 5.3** <0.7 <0.7 <0.7 38 no¹Shut-off temperature is defined as the lowest restrictive temperatureat which a 100-fold or greater reduction of plaque titer is observed(bold figures in table).*Small-plaque phenotype (<50% wild-type plaque size)**Pinpoint-plaque phenotype (<10% wild-type plaque size)

One of the mutant progeny had the small plaque phenotype, RSV cpsp143(sp refers to the small plaque (sp) phenotype), and the remaining mutantprogeny had the ts phenotype. The RSV cpts mutants exhibit a variationin ability to produce plaques in monolayer cultures in vitro over thetemperature range 37° C. to 40° C., with cpts368 retaining the abilityto produce plaques at 40° C., whereas the most temperature-sensitive(ts) virus, cpts248, failed to produce plaques at 38° C. Thus, severalof the mutagenized cpRSV progeny exhibit a marked difference from theircpRSV parent virus with respect to temperature-sensitivity of plaqueformation.

Replication and Genetic Stability Studies in Mice

The level of replication of the cpRSV derived mutants in the upper andlower respiratory tracts of BALB/c mice was studied next (Table 2). Itwas found that cpts530 and cpts248, two of the mutants exhibiting thegreatest temperature sensitivity (see Table 1), were about 7- to 12-foldrestricted in replication in the nasal turbinates of the mice (Table 2).However, none of the viruses was restricted in replication in the lungscompared to the cpRSV parent virus. This greater restriction ofreplication in the nasal turbinates than in the lungs is notcharacteristic of ts mutants, which generally are more restricted inreplication in the warmer lower respiratory tract (Richman and Murphy,Rev. Infect. Dis. 1:413-433 (1979). The virus produced in the lungs andnasal turbinates retained the ts character of the input virus (data notpresented). The present findings suggested that the combination of thets mutations on the background of the mutations of the cp parent virushas resulted in cpRSV ts progeny with a higher level of stability of thets phenotype after replication in vivo than had been seen withpreviously studied ts mutants.

To further explore the level of genetic stability of the ts phenotype ofthe cpRSV derived mutants, the efficiency of plaque formation of viruspresent in the lungs and nasal turbinates of nude mice was studied fortwo mutagenized cpRSV progeny that were among the most ts, namelycpts248 and cpts530. Nude mice were selected because they areimmunocompromised due to congenital absence of functional T-cells, and avirus can replicate for a much longer period of time in these hosts.This longer period of replication favors the emergence of virus mutantswith altered phenotype. The virus present on day 12 (NOTE: in normalmice, virus is no longer detectable at this time) was characterized andfound to retain an unaltered ts phenotype (Table 3). As expected, thets-1 mutant included in the test as a positive control exhibited anunstable ts phenotype in vivo. Thus, contrary to previous evaluation ofts mutant viruses in rodents, the results show that a high level ofstability of the ts phenotype of the cpRSV derived mutants followingprolonged replication in rodents was achieved, which represents asignificant and heretofore unattained very desirable property in theviruses of the invention. TABLE 2 Replication of cpts and cpsp- RSVmutants in BALB/c mice¹ Virus titer at 32° C. (mean log₁₀pfu/g tissuefrom the tissues of 8 animals ± standard error) Animals Shutoff Day 4Day 5 infected Temp. of Nasal Nasal with virus (° C.) Turbinates LungsTurbinates Lungs A2 >40 5.0 ± 0.16 5.8 ± 0.20 5.0 ± 0.11 5.8 ± 0.19wild-type cpRSV >40 4.7 ± 0.07 5.3 ± 0.18 4.8 ± 0.16 5.3 ± 0.21 ts-1 384.0 ± 0.19 4.7 ± 0.27 3.8 ± 0.33 4.9 ± 0.13 cpsp143 >40 4.5 ± 0.14 4.1 ±0.37 4.4 ± 0.39 4.6 ± 0.39 cpts368 40 4.8 ± 0.15 5.1 ± 0.35 4.7 ± 0.085.4 ± 0.23 cpts274 40 4.2 ± 0.19 5.0 ± 0.15 4.2 ± 0.11 5.1 ± 0.55cpts347 40 4.4 ± 0.32 4.9 ± 0.40 4.5 ± 0.33 5.2 ± 0.35 cpts142 39 4.1 ±0.34 5.0 ± 0.19 4.3 ± 0.24 5.8 ± 0.40 cpts299 39 3.9 ± 0.11 5.2 ± 0.153.9 ± 0.32 5.0 ± 0.29 cpts475 39 4.0 ± 0.18 5.3 ± 0.25 4.1 ± 0.23 4.9 ±0.42 cpts530 39 3.9 ± 0.18 5.3 ± 0.15 3.9 ± 0.14 5.3 ± 0.19 cpts248 383.9 ± 0.33 5.1 ± 0.29 4.2 ± 0.13 5.5 ± 0.35¹Mice were administered 10^(6.3) p.f.u. intranasally in a 0.1 mlinoculum on day 0, then sacrificed on day 4 or 5.

TABLE 3 The genetic stability of RSV cpts-248 and cpts-530 followingprolonged replication in nude mice Efficiency of plaque formation atindicated temperature of virus present in nasal turbinates (n.t.) orlungs of nude mice sacrificed 12 days after virus administration¹ 32° C.37° C. 38° C. 40° C. Mean titer Mean titer Mean titer Mean titer Tissue% (log₁₀pfu % % animals (log₁₀pfu % % animals (log₁₀pfu % % animals(log₁₀pfu harvest Num- animals per gram animals with virus per gramanimals with virus per gram animals with virus per gram or ber withtissue or with with tissue or with with tissue or with with tissue orAnimals input of virus ml virus altered ml virus altered ml virusaltered ml infected virus ani- detect- inocu- detect- ts inocu- detect-ts inocu- detect- ts inocu- with tested mals able lum)² able phenotypelum)² able phenotype lum)² able phenotype lum)² cpts-248 n.t. 19 100 3.8± 0.34 0 0 <2.0 0 0 <2.0 0 0 <2.0 ″ lungs ″  90 2.0 ± 0.29 0 0 <1.7 0 0<1.7 0 0 <1.7 cpts-530 n.t. 20 100 3.0 ± 0.26 0 0 <2.0 0 0 <2.0 0 0 <2.0″ lungs ″ 100 2.4 ± 0.29 0 0 <1.7 0 0 <1.7 0 0 <1.7 ts-1 n.t. 19 100 3.7± 0.23 74  74  2.7 ± 0.57 63  63  2.4 ± 0.36 10  10  2.0 ± 0.13 ″ lungs″ 100 2.5 ± 0.30 74  74  1.8 ± 0.21 35  32  1.8 ± 0.15 0 0 <1.7Efficiency cpts- — — 4.9 — <0.7 — <0.7 — <0.7 of plaque 248 formationcpts- — — 5.5 — 3.7* — <0.7 — <0.7 of input 530 viruses ts-1 — — 6.1 —3.3 — 2.7 — <0.7¹Plaque titers shown represent the mean log₁₀pfu/gram tissue of 19 or 20samples ± standard error.²Each animal received 10^(6.3) p.f.u. intranasally in a 0.1 ml inoculumof the indicated virus on day 0.*Small-plaque phenotype only.

In Chimpanzees

The level of attenuation of the cpRSV ts derivative was next evaluatedin the seronegative chimpanzee, a host most closely related to humans.Trials in chimpanzees or owl monkeys are conducted according to thegeneral protocol of Richardson et al., J. Med. Virol. 3:91-100 (1979);Crowe et al., Vaccine 11:1395-1404 (1993), which are incorporated hereinby reference. One ml of suspension containing approximately 10⁴plaque-forming units (PFU) of mutagenized, attenuated virus is givenintranasally to each animal. An alternate procedure is to inoculate theRSV into both the upper and lower respiratory tract at a dose of 10⁴ PFUdelivered to each site. Chimpanzees are sampled daily for 10 days, thenevery 3-4 days through day 20. The lower respiratory tract ofchimpanzees can be sampled by tracheal lavage according to the protocolof Snyder et al., J. Infec. Dis. 154:370-371 (1986) and Crowe et al.,Vaccine 11:1395-1404 (1993). Some animals are challenged 4 to 6 weekslater with the wild-type virus. Animals are evaluated for signs ofrespiratory disease each day that nasopharyngeal specimens are taken.Rhinorrhea is scored from 0 to 4+, with 2+ or greater being consideredas evidence of significant upper respiratory disease.

Virus is isolated from nasal and throat swab specimens and tracheallavage fluids by inoculation into RSV-sensitive HEp-2 cells as describedabove. Quantities of virus can also be determined directly by the plaquetechnique using HEp-2 cells as described in Schnitzer et al., J. Virol.17:431-438 (1976), which is incorporated herein by reference. Specimensof serum are collected before administration of virus and at 3 to 4weeks post-inoculation for determination of RSV neutralizing antibodiesas described in Mills et al., J. Immununol. 107:123-130 (1970), which isincorporated herein by reference.

The most ts and attenuated of the cpRSV derivative (cpts248) was studiedand compared to wild-type RSV and the cpRSV parent virus (Table 4).Replication of the cpRSV parent virus was slightly reduced in thenasopharynx compared to wild-type, there was a reduction in the amountof rhinorrhea compared to wild-type virus, and there was an approximate600-fold reduction in virus replication in the lower respiratory tractcompared to wild-type. Clearly, the cp virus was significantlyrestricted in replication in the lower respiratory tract of chimpanzees,a very desirable property not previously identified from priorevaluations of cpRSV in animals or humans. More significantly, the cpts248 virus was 10-fold restricted in replication in the nasopharynxcompared to wild-type, and this restriction was associated with a markedreduction of rhinorrhea. These findings indicated that the cpRSV derivedmutant possesses two highly desirable properties for a live RSV vaccine,namely, evidence of attenuation in both the upper and the lowerrespiratory tracts of highly susceptible seronegative chimpanzees. Thelevel of genetic stability of the virus present in the respiratory tractof chimpanzees immunized with cpts248 was evaluated next (Table 5). Thevirus present in the respiratory tract secretions retained the tsphenotype, and this was seen even with the virus from chimpanzee No. 3on day 8 that was reduced 100-fold in titer at 40° C. and exhibited thesmall plaque phenotype at 40° C., indicating that its replication wasstill temperature-sensitive. This represents the most genetically stablets mutant identified prior to the time of this test. The increasedstability of the ts phenotype of the cpts248 and cpts530 virusesreflects an effect of the cp mutations on the genetic stability of themutations that contribute to the ts phenotype in vivo. Thus, the tsmutations in the context of the mutations already present in the cp3131parent virus appear to be more stable than would be expected in theirabsence. This important property has not been previously observed orreported. Infection of chimpanzees with the cpts 248 induced a hightiter of serum neutralizing antibodies, as well as antibodies to the Fand G glycoproteins (Table 6). Significantly, immunization with cpts248protected the animals from wild-type RSV challenge (Table 7), indicatingthat this mutant functions as an effective vaccine virus in a host thatis closely related to humans.

These above-presented findings indicate that the cpts248 virus has manyproperties desirable for a live RSV vaccine, including: 1) attenuationfor the upper and lower respiratory tract; 2) increased geneticstability after replication in vivo, even after prolonged replication inimmunosuppressed animals; 3) satisfactory immunogenicity; and 4)significant protective efficacy against challenge with wild-type RSV.The cpts530 virus shares with cpts248 similar temperature sensitivity ofplaque formation, a similar degree of restriction of replication in thenasal turbinates of mice, and a high level of genetic stability inimmunodeficient nude mice, whereby it also represents an RS virusvaccine strain. TABLE 4 Replication of cpts-RSV 248, cp-RSV, orwild-type RSV A2 in the upper and lower respiratory tract ofseronegative chimpanzees Virus recovery Animal infected NasopharynxTrachea Rhinorrhea with indicated Route of Chimpanzee Duration^(b) Peaktiter Duration^(b) Peak titer score virus Inoculation number (days)(log₁₀pfu/ml) (days) (log₁₀pfu/ml) Mean^(c) Peak cpts-248 IN + IT 1 104.6  8^(d) 5.4 0.2 1 IN + IT 2 10 4.5  6 2.2 0.1 1 IN + IT 3  9 4.7 102.1 0.1 1 IN + IT 4  9 4.2  8^(d) 2.2 0.1 1 mean 9.5 mean 4.5 mean 8.0mean 3.0 mean 0.1 cp-RSV IN 5 20 5.3  8^(d) 2.9 1.0 3 IN 6 16 5.8  6^(d)3.0 1.8 3 IN + IT 7 13 4.3  6^(d) 3.0 0.6 1 IN + IT 8 16 5.0 10^(d) 2.80.5 1 mean 16 mean 5.1 mean 7.5 mean 2.9 mean 1.0 A2 wild-type IN  9  95.1 13 5.4 1.0 1 IN 10  9 6.0  8 6.0 1.7 4 IN + IT 11 13 5.3  8 5.9 2.13 IN + IT 12  9 5.4  8 5.6 1.0 3 mean 10 mean 5.5 mean 9.3 mean 5.7 mean1.4^(a)IN = Intranasal administration only, at a dose of 10⁴ p.f.u. in a1.0 ml inoculum; IN + IT = Both intranasal and intratrachealadministration, 10⁴ p.f.u. in a 1.0 ml inoculum at each site.^(b)Indicates last day post-infection on which virus was recovered.^(c)Mean rhinorrhea score represents the sum of daily scores for aperiod of eight days surrounding the peak day of virus shedding, dividedby eight. Four is the highest score; zero is the lowest score.^(d)Virus isolated only on day indicated.

TABLE 5 Genetic stability of virus present in original nasopharyngeal(NP) swabs or tracheal lavage (TL) specimens obtained from animalsexperimentally infected with cptsRSV 248. Virus Titer of RSV atindicated obtained on temperature (log₁₀pfu/ml) Chimpanzee NP swab orpost-infection Titer at Titer at Titer at number TL specimen day 32° C.39° C. 40° C. 1^(a) NP 3 3.2 <0.7 NT ″ 4 2.7 <0.7 NT ″ 5 4.2 <0.7 NT ″ 63.8 <0.7 NT ″ 7 4.6 <0.7 NT ″ 8 4.5 <0.7 NT ″ 9 2.6 <0.7 NT ″ 10 2.0<0.7 NT TL 6 5.4 <0.7 NT ″ 8 2.7 <0.7 NT 2^(a) NP 3 3.2 <0.7 NT ″ 4 3.7<0.7 NT ″ 5 4.5 <0.7 NT ″ 6 4.1 <0.7 NT ″ 7 3.3 <0.7 NT ″ 8 4.2 <0.7 NT″ 9 2.8 <0.7 NT ″ 10 1.6 <0.7 NT TL 6 2.2 <0.7 NT 3  NP 3 2.7 <0.7 <0.7″ 4 3.4 <0.7 <0.7 ″ 5 2.9 <0.7 <0.7 ″ 6 3.3 <0.7 <0.7 ″ 7 3.4 0.7^(b)<0.7 ″ 8 4.7 3.5^(b) 2.0^(c) ″ 9 1.9 <0.7 <0.7 TL 6 1.8 <0.7 <0.7 ″ 81.9 1.2^(b) <0.7 ″ 10 2.1 1.3^(b) <0.7 4  NP 3 3.2 <0.7 NT ″ 4 2.7 <0.7NT ″ 5 3.4 <0.7 NT ″ 6 3.3 <0.7 NT ″ 7 4.2 <0.7 NT ″ 8 3.5 <0.7 NT ″ 92.1 <0.7 NT TL 8 2.2 <0.7 NTNT = Not tested^(a)Isolates (once-passaged virus suspensions with average titerlog₁₀pfu/ml of 4.0) were generated from these chimpanzees from eachoriginal virus-containing nasopharyngeal swab specimen or tracheallavage specimen and tested for efficiency of plaque formation at 32°,39° and 40° C. No isolate was able to form plaques at 39° C. Isolatesfrom chimpanzees 3 and 4 were not tested in this manner.^(b)The percent titer at 39° C. versus that at 32° C.: NP swab day 7 =0.2%, NP swab day 8 = 6T, TL day 8-20%, TL day 10 = 16%. All plaqueswere of small-plaque phenotype only; no wild-type size plaques seen.^(c)The percent titer at 40° C. versus that at 32° C. was 0.2%. Allplaques were of pinpoint-plaque phenotype; wild-type size plaques werenot detected.

TABLE 6 Serum antibody responses of chimpanzees infected with RSVcpts-248, cp-RSV, or RSV A2 wild-type Serum antibody titers (reciprocalmean log₂) Animals Neutralizing ELISA-F ELISA-G immunized No. of Day DayDay Day Day Day with Chimpanzees 0 28 0 28 0 28 cpts-248 4 <3.3 10.7 7.315.3 6.3 9.8 cp-RSV 4 <3.3 11.2 11.3 15.3 9.3 12.3 RSVA2 4 <3.3 11.2 8.315.3 7.3 10.3 wild-type

TABLE 7 Immunization of chimpanzees with cpts-248 induces resistance toRSV A2 wild-type virus challenge on day 28 Response to challenge with10⁴ p.f.u. wild-type virus administered on day 28 Serum neutralizingantibody titer (reciprocal log₂) Virus Recovery on Nasopharynx TracheaRhinorrhea day indicated Virus used to Chimpanzee Duration Peak titerDuration Peak titer score Day 42 immunize animal number (days)(log₁₀pfu/ml) (days) (log₁₀pfu/ml) Mean* Peak Day 28 or 56 cpts-248 1 52.7 0 <0.7 0 0 10.1 11.0 2 9 1.8 0 <0.7 0 0 10.3 14.5 cp-RSV 5 5 1.0 0<0.7 0 0 11.1 13.3 6 8 0.7 0 <0.7 0 0 11.4 12.9 none 9 9 5.1 13 5.4 1.01 <3.3 12.4 10 9 6.0 8 6.0 1.7 4 <3.3 13.2 11 13 5.3 8 5.9 2.1 3 <3.311.6 12 9 5.4 8 5.6 1.0 3 <3.3 11.2*Mean rhinorrhea score represents the sum of scores during the eightdays of peak virus shedding divided by eight. Four is the highest score.A score of zero indicates no rhinorrhea detected on any day of theten-day observation period.

Further Attenuations

Since RS virus produces more symptoms of lower respiratory tract diseasein human infants than in the 1-2 year old chimpanzees used in theseexperimental studies, and recognizing that mutants which aresatisfactorily attenuated for the chimpanzee may not be so forseronegative infants and children, the cpts248 and 530 derivatives,which possess the very uncharacteristic ts mutant properties ofrestricted replication and attenuation in the upper respiratory tractand a higher level of genetic stability, were further mutagenized.

Progeny viruses that exhibited a greater degree oftemperature-sensitivity in vitro than cpts248 or that had the smallplaque phenotype were selected for further study. Mutant derivatives ofthe cpts248 that possessed one or more additional ts mutations wereproduced by 5-fluorouracil mutagenesis (Table 8). Ts mutants that weremore temperature-sensitive (ts) than the cpts248 parental strain wereidentified, and some of these had the small plaque (sp) phenotype. Thesecpts248 derivatives were administered to mice. cpts248/804, 248/955,248/404, 248/26, 248/18, and 248/240 mutants were more restricted inreplication in the upper and lower respiratory tract of the mouse thantheir cpts248 parental virus (Table 9). Thus, viable mutants of cpts248which were more attenuated than their cpts248 parental virus wereidentified, and these derivatives of cpts248 exhibited a wide range ofreplicative efficiency in mice, with cpts248/26 being the mostrestricted. The ts phenotype of the virus present in nasal turbinatesand lungs of the mice was almost identical to that of the input virus,indicating genetic stability. A highly attenuated derivative of cpts248,the cpts248/404 virus, was 1000-fold more restricted in replication inthe nasopharynx compared to wild-type. The cpts248/404 mutant,possessing at least three attenuating mutations, was also highlyrestricted in replication in the upper and lower respiratory tracts offour seronegative chimpanzees and infection did not induce rhinorrhea(Table 10). Again, this virus exhibited a high degree of reduction inreplication compared to wild-type, being 60,000-fold reduced in thenasopharynx and 100,000-fold in the lungs. Nonetheless, two chimpanzeeswhich were subsequently challenged with RSV wild-type virus were highlyresistant (Table 11).

Five small-plaque mutants of cpts248/404 were derived by chemicalmutagenesis in a similar fashion to that described above. Suspensions ofonce-amplified plaque progeny were screened for the small-plaque (sp)phenotype by plaque titration at 32° C. on HEp-2 cells, and workingsuspensions of virus were prepared as described above.

Five of the plaque progeny of the mutagenized cpts248/404 virusexhibited a stable sp phenotype. The shut-off temperature of each mutantwas 35° C. or less (Table 12), suggesting that each of these spderivatives of the cpts248/404 virus also had acquired an additional tsmutation. Following intranasal inoculation of Balb/c mice with 10^(6.3)p.f.u. of a sp derivative of the cpts248/404, virus could not bedetected in the nasal turbinates of mice inoculated with any of these spderivatives. However, virus was detected in low titer in the lungs inone instance. These results indicate >300-fold restriction ofreplication in the nasal turbinates and >10,000-fold restriction inlungs compared with wild-type RSV.

Further ts derivatives of the cpts530 virus were also generated (Table13). As with the cpts248 derivatives, the cpts-530 derivatives were morerestricted in replication in mice than the cpts530 parental strain. Onemutant, cpts-530/1009, was 30 times more restricted in replication inthe nasal turbinates of mice. This cpts530 derivative, is also highlyrestricted in replication in the upper and lower respiratory tract ofseronegative chimpanzees (Table 14). In the nasopharynx, cpts530 was30-fold restricted in replication, while cpts530/1009 was 100-foldrestricted compared to wild-type virus. Both of the cpts mutants werehighly restricted (20,000 to 32,000-fold) in the lower respiratory tractcompared with wild-type virus, even when the mutants were inoculateddirectly into the trachea. Also, chimpanzees previously infected withcpts530/1009, cpts530 or cpRSV exhibited significant restriction ofvirus replication in the nasopharynx and did not develop significantrhinorrhea following subsequent combined intranasal and intratrachealchallenge with wild-type RSV (Table 15). In addition, chimpanzeespreviously infected with any of the mutants exhibited completeresistance in the lower respiratory tract to replication of wild-typechallenge virus.

These results were completely unexpected based on experience gainedduring prior studies. For example, the results of an earlier studyindicated that the in vivo properties of RSV ts mutants derived from asingle cycle of 5-fluorouracil mutagenesis could not be predicted apriori.

Moreover, although one of the first four ts mutants generated in thismanner exhibited the same shut off temperature for is plaque formationas the other mutants, it was overattenuated when tested in susceptiblechimpanzees and susceptible infants and young children (Wright et al.,Infect Immun. 37 (1):397-400 (1982). This indicated that the acquisitionof the ts phenotype resulting in a 37-38° C. shut off temperature forplaque formation did not reliably yield a mutant with the desired levelof attenuation for susceptible chimpanzees, infants and children.Indeed, the results of studies with heretofore known ts mutantscompletely fail to provide any basis for concluding that introduction ofthree independent mutations (or sets of mutations) into RSV bycold-passage followed by two successive cycles of chemical mutagenesiscould yield viable mutants which retain infectivity for chimpanzees (andby extrapolation, young infants) and exhibit the desired level ofattenuation, immunogenicity and protective efficacy required of a livevirus vaccine to be used for prevention of RSV disease.

The above-presented results clearly demonstrate that certain tsderivatives of the cpRSV of the invention have a satisfactory level ofinfectivity and exhibit a significant degree of attenuation for mice andchimpanzees. These mutant derivatives are attenuated and appear highlystable genetically after replication in vivo. These mutants also inducesignificant resistance to RSV infection in chimpanzees. Thus, thesederivatives of cpRSV represent virus strains suitable for use in a liveRSV vaccine designed to prevent serious human RSV disease. TABLE 8 Theefficiency of plaque formation of ten mutants derived from RSV cpts248by additional 5FU mutagenesis. The titer of virus (log₁₀pfu/ml) ShutoffSmall- at the indicated temperature (° C.) temperature plaques Virus 3235 36 37 38 39 40 (° C.)¹ at 32 C. A2 wild-type 4.5 4.6 4.4 4.5 4.5 3.83.8 >40 no cpRSV 4.7 4.4 4.3 4.3 4.2 3.7 3.5 >40 no ts-1 5.6 5.4 4.9 4.42.7 2.0 <0.7 38 no cpts-248 3.4 3.0 2.6* 1.7** <0.7 <0.7 <0.7 38 no248/1228 5.5* 5.3* 5.3** <0.7 <0.7 <0.7 <0.7 37 yes 248/1075 5.3* 5.3*5.1** <0.7 <0.7 <0.7 <0.7 37 yes 248/965 4.5 4.2 4.2* <0.7 <0.7 <0.7<0.7 37 no 248/967 4.4 3.7 3.6* <0.7 <0.7 <0.7 <0.7 37 no 248/804 4.94.5 4.0* <0.7 <0.7 <0.7 <0.7 37 no 248/955 4.8 3.7 2.8* <0.7 <0.7 <0.7<0.7 36 no 248/404 3.6 2.9* <0.7 <0.7 <0.7 <0.7 <0.7 36 no 248/26 3.12.9* <0.7 <0.7 <0.7 <0.7 <0.7 36 no 248/18 4.0* 4.0** <0.7 <0.7 <0.7<0.7 <0.7 36 yes 248/240 5.8* 5.7** <0.7 <0.7 <0.7 <0.7 <0.7 36 yes¹Shut-off temperature is defined as the lowest restrictive temperatureat which a 100-fold or greater reduction of plaque titer in Hep-2 cellsis observed (bold figures in table).*Small-plaque phenotype (<50% wild-type plaque size).**Pinpoint-plaque phenotype (<10% wild-type plaque size).

TABLE 9 Replication and genetic stability of ten mutants derived fromRSV cpts-248 in Balb/c mice¹ Shutoff temperature Virus titer (meanlog₁₀pfu/g tissue of six animals ± standard error) Virus used to ofNasal turbinates Lungs infect animal virus (° C.) 32° C. 36° C. 37° C.38° C. 32° C. 36° C. 37° C. 38° C. A2 wild-type >40 5.1 ± 0.15 5.2 ±0.23 5.2 ± 0.14 5.2 ± 0.27 6.1 ± 0.14 5.8 ± 0.23 6.0 ± 0.12 5.9 ± 0.17cp-RSV >40 4.9 ± 0.20 5.1 ± 0.16 4.9 ± 0.24 4.9 ± 0.22 6.0 ± 0.16 5.9 ±0.23 5.6 ± 0.15 5.6 ± 0.13 ts-1 38 3.9 ± 0.25 2.7 ± 0.27 2.4 ± 0.42 2.5± 0.29 4.1 ± 0.21 3.5 ± 0.23 2.6 ± 0.18 2.0 ± 0.23 cpts-248 38 4.0 ±0.16 2.5 ± 0.34 <2.0 <2.0 4.4 ± 0.37 1.8 ± 0.15 <1.7 <1.7 248/1228 374.1 ± 0.15 2.4 ± 0.48 <2.0 <2.0 2.0 ± 0.37 <1.7 <1.7 <1.7 248/1075 374.2 ± 0.18 2.4 ± 0.40 <2.0 <2.0 5.5 ± 0.16 3.5 ± 0.18 <1.7 <1.7 248/96537 3.8 ± 0.23 <2.0 <2.0 <2.0 4.5 ± 0.21 3.4 ± 0.16 <1.7 <1.7 248/967 374.4 ± 0.20 <2.0 <2.0 <2.0 5.4 ± 0.20 3.6 ± 0.19 <1.7 <1.7 248/804 37 2.9± 0.19 <2.0 <2.0 <2.0 3.6 ± 0.19 <1.7 <1.7 <1.7 248/955 36 3.2 ± 0.10<2.0 <2.0 <2.0 3.2 ± 0.22 <1.7 <1.7 <1.7 248/404 36  2.1 ± 0.31² <2.0<2.0 <2.0  4.4 ± 0.12² 1.8 ± 0.20 <1.7 <1.7 248/26 36 <2.0 <2.0 <2.0<2.0 2.3 ± 0.20 <1.7 <1.7 <1.7 248/18 36 2.9 ± 0.99 <2.0 <2.0 <2.0 4.3 ±0.23 1.8 ± 0.15 <1.7 <1.7 248/240 36 2.9 ± 0.82 <2.0 <2.0 <2.0 3.9 ±0.12 <1.7 <1.7 <1.7¹Mice were administered 10^(6.3) p.f.u. intranasally under lightanesthesia on day 0, then sacrificed by CO₂ asphyxiation on day 4.²In a subsequent study, the level of replication of the cpts-248/404virus was found to be 2.4 ± 0.24 and 2.6 ± 0.31 in the nasal turbinatesand lungs, respectively.

TABLE 10 Replication of cpts-RSV 248/404, cpts-RSV 248/18, cpts-RSV 248,cp-RSV, or wild-type RSV A2 in the upper and lower respiratory tract ofseronegative chimpanzees Virus recovery Nasopharynx Trachea RhinorrheaAnimal infected Route of Chimpanzee Duration^(b) Peak titer Duration^(b)Peak titer scores with indicated virus Inoculation number (days)(log₁₀pfu/ml) (days) (log₁₀pfu/ml) Mean^(c) Peak cpts-248/404 IN + IT 130 <0.7  0 <0.7 0   0 IN + IT 14 0 <0.7  0 <0.7 0   0 IN + IT 15 8 1.9  0<0.7 0.3 2 IN + IT 16 9 2.0  0 <0.7 0.2 1 mean 4.3 mean 1.3 mean 0 mean<0.7 mean 0.1 mean 0.8 cpts-248# IN + IT 1 10 4.6  8^(d) 5.4 0.2 1 IN +IT 2 10 4.5  6 2.2 0.1 1 IN + IT 3 9 4.7 10 2.1 0.1 1 IN + IT 4 9 4.2 8^(d) 2.2 0.1 1 mean 9.5 mean 4.5 mean 8.0 mean 3.0 mean 0.1 mean 1.0cp-RSV# IN 5 20 5.3  8^(d) 2.9 1.0 3 IN 6 16 5.8  6^(d) 3.0 1.8 3 IN +IT 7 13 4.3  6^(d) 3.0 0.6 1 IN + IT 8 16 5.0 10^(d) 2.8 0.5 1 mean 16mean 5.1 mean 7.5 mean 2.9 mean 1.0 mean 2.0 A2 wild-type# IN 9 9 5.1 135.4 1.0 1 IN 10 9 6.0  8 6.0 1.7 4 IN + IT 11 13 5.3  8 5.9 2.1 3 IN +IT 12 9 5.4  8 5.6 1.0 3 mean 10 mean 5.5 mean 9.3 mean 5.7 mean 1.4mean 2.8^(a)IN Intranasal administration only; IN + IT = Both intranasal andintratracheal administration.^(b)Indicates last day post-infection on which virus was recovered.^(c)Mean rhinorrhea score represents the sum of daily scores for aperiod of eight days surrounding the peak day of virus shedding, dividedby eight. Four is the highest score; zero is the lowest score.^(d)Virus isolated only on day indicated.#These are the same animals included in Tables 4 and 7.

TABLE 11 Immunization of chimpanzees with cpts-248/404 inducesresistance to RSV A2 wild-type virus challenge on day 28 Serumneutralizing antibody titer Virus Recovery (reciprocal log₂) onNasopharynx Tracheal lavage day indicated^(b) Virus used to ChimpanzeeDuration Peak titer Duration Peak titer Rhinorrhea scores Day 49 orimmunize animal number (days) (log₁₀pfu/ml) (days) (log₁₀pfu/ml)Mean^(a) Peak Day 28 56 cpts-248/404 13 0 <0.7 0 <0.7 0 0 7.9 9.0 14 83.4 0 <0.7 0 0 7.0 12.5 mean 4.0 mean 2.0 mean 0 mean <0.7 mean 0 mean 0mean 7.5 mean 10.8 cpts-248# 1 5 2.7 0 <0.7 0 0 11.5 13.0 2 9 1.8 0 <0.70 0 12.7 14.5 mean 7.0 mean 2.3 mean 0 mean <0.7 mean 0 mean 0 mean 12.1mean 13.8 cp-RSV# 5 5 1.0 0 <0.7 0 0 12.2 11.1 6 8 0.7 0 <0.7 0 0 11.99.9 mean 6.5 mean 0.9 mean 0 mean 0.7 mean 0 mean 0 mean 12.1 mean 10.5None 9 9 5.1 13 5.4 1.0 1 <3.3 11.0 10 9 6.0 8 6.0 1.7 4 <3.3 9.8 11 135.3 8 5.9 2.1 3 <3.3 9.4 12 9 5.4 8 5.6 1.0 3 <3.3 14.5 mean 10 mean 5.5mean 9.2 mean 5.7 mean 1.4 mean 2.8 mean <3.3 mean 11.2^(a)Mean rhinorrhea score represents the sum of scores during the eightdays of peak virus shedding divided by eight. Four is the highest score.A score of zero indicates no rhinorrhea detected on any day of theten-day observation period.#These are the same animals included in Tables 4, 7 and 10.^(b)Serum neutralizing titers in this table, including those fromanimals previously described, were determined simultaneously in oneassay.

TABLE 12 The efficiency of plaque formation and replication of Balb/cmice of five small-plaque derivatives of RSV cpts-248/404. Efficiency ofplaque formation tested in HEp-2 cells at permissive and restrictivetemperatures Replication The titer of virus (log₁₀pfu/ml) Shut-offSmall- in Balb/c mice² at the indicated temperature (° C.) temp plaquesNasal Virus 32 35 36 37 38 39 40 (° C.)¹ at 32° C. turbinates³ Lungs³ A2wild-type 6.0 6.1 6.0 5.8 5.9 5.4 5.4 >40 no 4.5 ± 0.34 5.6 ± 0.13cp-RSV 6.2 6.2 6.0 6.0 5.9 5.6 5.4 >40 no 4.5 ± 0.10 5.3 ± 0.20 cpts-2487.5 7.3 6.2**  5.3** <0.7 <0.7 <0.7 37 no 3.3 ± 0.35 4.8 ± 0.14 248/4045.5  3.6** <0.7  <0.7  <0.7 <0.7 <0.7 36 no 2.4 ± 0.24 2.6 ± 0.31248/404/774 2.9* <0.7  <0.7  <0.7  <0.7 <0.7 <0.7 ≦35 yes <2.0 1.8 ±0.24 248/404/832 5.5** <0.7  <0.7  <0.7  <0.7 <0.7 <0.7 ≦35 yes <2.0<1.7 248/404/886 5.0** <0.7  <0.7  <0.7  <0.7 <0.7 <0.7 ≦35 yes <2.0<1.7 248/404/893 5.4** <0.7  <0.7  <0.7  <0.7 <0.7 <0.7 ≦35 yes <2.0<1.7 248/404/ 4.4*  2.2** <0.7 <0.7  <0.7  <0.7 <0.7 35 yes <2.0 <1.71030¹Shut-off temperature is defined as the lowest restrictive temperatureat which at 100-fold or greater reduction of plaque titer is observed(bold figures in table).²Mice were administered 10^(6.3) p.f.u. intranasally under lightanesthesia on day 0, then sacrificed by CO₂ asphyxiation on day 4 whentissues were harvested for virus titer.³Mean log₁₀pfu/g tissue of six animals ± standard error.*Small-plaque phenotype (<50% wild-type plaque size).**Pinpoint-plaque phenotype (<10% wild-type plaque size).

TABLE 13 The efficiency of plaque formation and level of replication inmice of 14 mutants derived from RSV cpts530, compared with controlsReplication in mice² (mean log₁₀ In vitro efficiency of plaque formationpfu/g tissue of The titer of virus (log₁₀pfu/ml) at the indicatedShut-off six animals ± SE) temperature (° C.) Temp. Nasal RSV 32 34 3536 37 38 39 40 (° C.)¹ turbinates Lungs A2 6.3 6.3 6.1 6.2 6.3 6.3 6.15.6 >40 5.0 ± 0.14 5.8 ± 0.05 cpRSV 6.5 6.2 6.2 6.2 6.1 6.0 6.1 5.6 >40n.d. n.d cpts248 6.3 6.3 6.3 6.3  3.7** <0.7 <0.7 <0.7 37/38 4.1 ± 0.085.1 ± 0.13 248/404 6.3  5.7*  4.3** <0.7  <0.7  <0.7 <0.7 <0.7 35/36 2.1± 0.19 3.6 ± 0.10 cpts530 6.2 6.3 6.2 6.1  6.2* 5.5** <0.7 <0.7 39 3.4 ±0.09 4.3 ± 0.14 530/346 5.9 5.9 5.7 4.7 3.5 <0.7 <0.7 <0.7 37 3.3 ± 0.114.7 ± 0.09 530/977 5.0 4.4 3.6 3.4  2.8* <0.7 <0.7 <0.7 37 3.4 ± 0.112.7 ± 0.05 530/9 6.0 5.6 5.0  3.5*  3.5* <0.7 <0.7 <0.7 36 2.1 ± 0.063.5 ± 0.08 530/1009 4.8 4.0  3.7*  2.0**  1.5** <0.7 <0.7 <0.7 36 2.2 ±0.15 3.5 ± 0.13 530/667 5.5 4.9  4.5*  2.0** 0.7 <0.7 <0.7 <0.7 36 2.4 ±0.12 2.9 ± 0.15 530/1178 5.7 4.0 5.5  3.7**  2.0** <0.7 <0.7 <0.7 36 3.3± 0.06  42 ± 0.11 530/464 6.0  5.0*  4.7*  1.8** <0.7  <0.7 <0.7 <0.7 36<2.0 2.6 ± 0.10 530/403 5.7 5.1 4.3 2.9 <0.7  <0.7 <0.7 <0.7 36 <2.0<1.7 530/1074 5.1 4.6  4.1* <0.7  <0.7  <0.7 <0.7 <0.7 36 3.0 ± 0.13 3.8± 0.13 530/963 5.3 5.0  4.2* 0.7 <0.7  <0.7 <0.7 <0.7 36 2.0 ± 0.05 <1.7530/653 5.4 5.1 4.5 <0.7  <0.7  <0.7 <0.7 <0.7 36 2.2 ± 0.10 3.1 ± 0.16530/1003 5.6 4.1 2.5  2.1** <0.7  <0.7 <0.7 <0.7 35 <2.0 <1.7 530/10304.3  3.7*  1.7** <0.7  <0.7  <0.7 <0.7 <0.7 35 <2.0 1.8 ± 0.13 530/1885.0*  1.0* 1.0 <0.7  <0.7  <0.7 <0.7 <0.7 ≦34 <2.0 <1.7n.d. = not done*Small-plaque phenotype (<50% wild-type plaque size)**Pinpoint-plaque phenotype (<10% wild-type plaque size)¹Shut-off temperature is defined as the lowest restrictive temperatureat which a 100-fold or greater reduction of plaque titer is observed(bold figures in table).²Mice were administered 10^(6.3) p.f.u. intranasally under lightanesthesia on day 0, then sacrificed by CO₂ asphyxiation on day 4.

TABLE 14 Replication of cpts-530/1009, cp-RSV, or wild-type RSV A2 inthe upper and lower respiratory tract of seronegative chimpanzeesinduces serum neutralizing antibodies. Animals Virus Replication Day 28Infected with Trachea reciprocal 10⁴ pfu of Nasopharynx Peak Rhinorrheaserum indicated Route of Chimpanzee Duration^(b) Peak Titer Duration^(b)Titer Scores neutralizing virus Inoculation Number (days) (log₁₀pfu/ml)(days) (log₁₀pfu/ml) Mean^(c) Peak antibody titer^(g) cpts- IN + IT  1 93.1  0 <1.0 0.5 2 1,097 530/1009 IN + IT  2 10 4.0  10^(a) 1.8 0.5 2 416IN + IT  3 9 4.0  0 <1.0 0.8 2 1,552 IN + IT  4 9 3.3  0 <1.0 0.4 11,176 mean 9.3 mean 3.6 mean 2.5 mean 1.2 mean 0.5 mean 1.3 mean 1,060cpts-530 IN + IT  5 9 3.5   4^(e) 2.6 0.3 1 10,085 IN + IT  6 9 5.2  0<1.0 1.1 3 3,566 IN + IT  7 8 3.3  0 <1.0 0.6 2 588 IN + IT  8 8 4.4  0<1.0 0.5 2 1,911 mean 8.5 mean 4.1 mean 1.0 mean 1.4 mean 0.6 mean 2.0mean 4,038 cp-RSV IN   9^(d) 20 5.3   8^(e) 2.9 1.0 3 416 IN  10^(d) 165.8   6^(e) 3.0 1.8 3 2,048 IN + IT  11^(d) 13 4.3   6^(e) 3.0 0.6 1 776IN + IT  12^(d) 16 5.0  10^(e) 2.8 0.5 1 891 mean 16 mean 5.1 mean 7.5mean 2.9 mean 1.0 mean 2.0 mean 1,033 A2 wild-type IN  13^(f) 9 5.1 135.4 1.0 1 1,351 IN  14^(f) 9 6.0  8 6.0 1.7 4 676 IN + IT  15^(d) 13 5.3 8 5.9 2.1 3 1,261 IN + IT  16^(d) 9 5.4  8 5.6 1.0 3 20,171 mean 10mean 5.5 mean 9.3 mean 5.7 mean 1.4 mean 2.8 mean 5,865^(a)IN = Intranasal only; IN + IT = Both intranasal and intratrachealadministration.^(b)Indicates last day post-infection on which virus was recovered.^(c)Mean rhinorrhea score represents the sum of daily scores for aperiod of eight days surrounding the peak day of virus shedding, dividedby eight. Four is the highest score; zero is the lowest score.^(d)Animals from Crowe, et al., Vaccine 12: 691-699 (1994).^(e)Virus isolated only on day indicated.^(f)Animals from Collins, et al., Vaccine 8: 164-168 (1990).^(g)Determined by complement-enhanced 60% plaque reduction of RSV A2 inHEp-2 cell monolayer cultures. All titers were determined simultaneouslyin a single assay. The reciprocal titer of each animal on day 0 was <10.

TABLE 15 Immunization of chimpanzees with cpts-530/1009 or cpts-530induces resistance to wild-type RSV A2 virus challenge on day 28 Serumneutralizing Virus replication antibody Nasopharynx Tracheal lavageRhinorrhea (reciprocal log₂) Virus used for Chimpanzee Duration Peaktiter Duration Peak titer scores on day indicated^(d) immunizationnumber (days) (log₁₀pfu/ml) (days) (log₁₀pfu/ml) Mean^(a) Peak Day 28Day 49 or 56 cpts-530/1009 3 7 2.1 0 <0.7 0 0 1,552 3,823 4 0 <0.7 0<0.7 0 0 1,176 1,911 cpts-530 5 0 <0.7 0 <0.7 0 0 10,085 6,654 6 0 <0.70 <0.7 0.3 2 3,566 1,911 cp-RSV 11^(b) 5 1.0 0 <0.7 0 0 776 2,048 12^(b)8 0.7 0 <0.7 0 0 891 1,783 None 13^(b) 9 5.1 13 5.4 1.0 1 <10 1,35114^(b) 9 6.0 8 6.0 1.7 4 <10 676 15^(c) 13 5.3 8 5.9 2.1 3 <10 1,26116^(c) 9 5.4 8 5.6 1.0 3 <10 20,171^(a)Mean rhinorrhea scores represent the sum of scores during the eightdays of peak virus shedding divided by eight. Four is the highest score.^(b)Animals from Crowe et al. Vaccine 12: 691-699 (1994).^(c)Animals from Collins et al. Vaccine 8: 164-168 (1990).^(d)Serum neutralizing titers in this table, including those fromanimals previously described, were determined simultaneously in oneassay.

Effect of Passively-Acquired Serum RSV Antibodies on cpts Mutants inChimpanzees

In order to examine the effect of passively-acquired serum RSVantibodies on attenuation, immunogenicity and protective efficacy ofvarious cpts mutants of the invention in chimpanzees, the in vivoreplication of cpts248, cpts248/404, and cpts530/1009, was evaluated inseronegative chimpanzees which were infused with RSV immune globulin twodays prior to immunization (Table 16). Antibody was passivelytransferred in order to simulate the conditions which obtain in younginfants who possess maternally-derived RSV antibodies. In this way, itwas possible to assess the immunogenicity of each indicated mutant inthe presence of passive RSV antibodies to determine whether thereplication of highly attenuated viruses might be so reduced in infantswith a moderate to high titer of passive antibodies as to preclude theinduction of a protective immune response. It would also be possible todefine the nature of the antibody response to immunization in thepresence of passively acquired antibodies, and to define the extent andfunctional activity of the antibody response to virus challenge. Thelevel of virus replication in the nasopharynx and the associatedclinical score for the attenuated mutants was either not altered or onlymoderately altered by the presence of serum RSV antibodies when theinfection of those animals was compared to that of non-infusedseronegative chimpanzees. In contrast, the presence ofpassively-acquired antibodies effectively prevented virus replication ofcpts248 in the lower respiratory tract. Because the other two mutantswere already highly restricted in the lungs, the similar effect ofpassive antibodies could not be evaluated against those mutants.

Infusion of human RSV immune globulin yielded moderately high serumlevels of RSV F antibodies (titer 1:640 to 1:1600), and neutralizingantibodies (titer 1:199 to 1:252), but not appreciable amounts of serumRSV G antibody detectable above background (Table 17). Chimpanzees whowere infused with human RSV antibodies prior to immunization withcpts248/404, cpts530/1009, or cpts248 developed only one-tenth thequantity of RSV F antibodies and about one-half the titer ofneutralizing antibodies by day 42 post-immunization, compared tonon-infused immunized animals tested 28 days post-immunization. Becausethe infused human IgG contained substantial amounts of RSV F and RSVneutralizing antibodies, the residual antibodies from the infusionpresent in the 42-day serum samples could not be distinguished fromantibodies produced de novo in response to immunization. Given thehalf-life of human serum IgG antibodies in chimpanzees (Prince et al.,Proc. Natl. Acad. Sci. USA 85:6944-6948), the observed levels of F andneutralizing antibodies on day 42 following immunization with cpts arehigher than would be predicted for a residuum of the infusion. Inaddition, the RSV G antibody response following immunization of theinfused animals confirms that these chimpanzees mounted an immuneresponse to immunization.

Four to six weeks following immunization the chimpanzees were challengedwith wild-type RSV. Each of the animals exhibited complete resistance intheir lower respiratory tract, whether or not human IgG was infused twodays before immunization (Table 18). Non-infused animals developed amodest neutralizing antibody response to challenge or none at all (Table17). In contrast, the infused chimpanzees uniformly developed anunusually high titer of RSV neutralizing antibodies in response towild-type virus challenge despite the fact that virus replication hadbeen severely restricted (Tables 17 and 18). Moreover, followingimmunization in the presence of antibodies the most attenuated virus,cpts248/404, which exhibited the lowest level of virus replication andimmunization, had the highest post-challenge neutralizing antibodytiters (Table 17). In contrast, the least attenuated virus, cpts248, hadthe lowest post-challenge neutralizing antibody titer of the threegroups of infused animals. In addition to an increase in the quantity ofthe antibodies induced by immunization in the presence of antibodies,the quality of the antibodies, as measured by the neutralizing to ELISAF antibody titer ratio, was significantly greater than that induced byimmunization in seronegative animals (Table 17). The neutralizing/ELISAF ratio of the antibodies produced in the infused immunized animalspost-challenge was about 10- to 20-fold higher than in the non-infusedanimals and was consistent in all groups, regardless of mutant used toimmunize (Table 17).

The presence of passively-acquired antibodies at the time ofimmunization with a live virus vaccine might alter the immune responseto vaccine in three distinct ways. First, a significant decrease in thelevel of replication of vaccine virus might occur that results indecreased immunogenicity. It is possible that the passively-transferredRSV antibodies could restrict the replication of the vaccine viruses,especially the most defective mutants, and greatly decrease theirimmunogenicity. The results presented herein indicate that replicationof the least attenuated mutant (cpts248) in the lower respiratory tractwas indeed abrogated by the presence of passively-acquired serum IgG RSVantibodies, whereas replication in the upper respiratory tract did notappear to be significantly affected. The replication of the leastattenuated mutant tested, cpts248, was ≧200-fold more (i.e. completely)restricted in the lower respiratory tract in the presence of antibodies.The level of replication of the more attenuated mutants, cpts530/1009and cpts248/404, in the lower respiratory tract was highly restrictedeven in the seronegative animals. Therefore, a significant effect ofpassive antibodies on virus replication could not be detected.Immunization with each of the three attenuated mutants induced a highdegree of protection against wild-type challenge in both the upper andlower respiratory tracts, whether or not passively-acquired RSVantibodies were present at the time of immunization. Thus, the level ofreplication of the vaccine viruses in the upper respiratory tract ofpassively-immunized chimpanzees was sufficient to induce a high level ofresistance to wild-virus challenge which was comparable to that inducedin non-infused animals.

Second, passive antibodies can alter the immune response to infection bycausing a decrease in the amount and functional activity of antibodiesthat are induced. For this reason the magnitude and the character of theantibody response to live virus immunization in the presence of passiveantibodies was analyzed. Postimmunization serum ELISA IgG F antibodytiters of immunized, infused animals were 10-fold lower than thepostimmunization F titers of non-infused seronegative animals. The serumRSV neutralizing antibody response was also slightly decreased in thoseanimals, on average being 2-fold lower than in non-infused animals.Because some of the ELISA F and neutralizing antibodies detectedpostimmunization represent residual antibodies from the infusion, theactual decrease of the neutralizing and F antibody response caused bypreexisting antibodies is probably even more significant than isapparent. Moreover, the human immune globulin preparation used containeda low level of antibodies to the G glycoprotein of RSV (Table 17). Thispetted an examination of the IgG RSV G glycoprotein antibody response ofthe chimpanzees to infection with the candidate vaccine viruses. The Gantibody responses demonstrated at least a 4-fold or greater increase,indicating that each of the passively-immunized animals was infected byvaccine virus, including chimpanzees immunized with cpts248/404 whichdid not shed virus. The magnitude of the G antibody response toimmunization did not appear to be adversely influenced by the passivelytransferred antibodies.

Thirdly, the antibody response to RSV wild-type virus challenge ofanimals immunized in the presence of passively-acquired antibodies couldbe altered. Chimpanzees immunized in the absence of infused antibodiesexhibited significant resistance to subsequent RSV challenge. Inaddition, these animals failed to develop an appreciable antibodyresponse to challenge virus. Although each of the 6 infused, immunizedanimals also exhibited significant resistance to RSV, a greatly enhancedantibody response to challenge was observed. Post-challenge F or Gantibody levels in the treated animals immunized with cpts530/1009 orcpts248/404 were increased at least 10-fold, while the neutralizingantibody response represented as much as an 800-fold increase. Theseresults suggest that repeated immunization of infants possessingmaternal antibodies with live attenuated mutants beginning very early inlife might stimulate effective resistance and an associated enhancedsecondary antibody response of high quality. The mechanism responsiblefor an enhanced immune response to second infection in the absence ofappreciable replication of the challenge virus is not understood. Thepresence of serum antibodies at the time of immunization, while allowinga modest antibody response to immunization in infused animals, favorsthe development of a B cell repertoire that elaborates antibodies ofhighly functional activity following subsequent RSV challenge.

The results reported herein are highly significant in that for the firsttime live attenuated RSV virus vaccine has been shown to be efficaciousin an animal model which mimics the target population for an RSVvaccine, i.e. the four to six week old infant having passively acquiredRSV neutralizing antibodies as a result of transplacental transfer fromthe mother. The importance of this finding is clear from the fact that,as discussed, supra, the high expectation that the passively transferredRSV antibodies would have inhibited the replication of the cpts vaccine,rendering it non-immunogenic and non-protective has, surprisingly, notbeen borne out. TABLE 16 Replication of RSV cpts-248/404, cpts-248, orcpts-530/1009 in the upper and lower respiratory tract of seronegativechimpanzees, some of which were infused with RSV neutralizing antibodiestwo days prior to immunization. Reciprocal serum Virus Replication RSVneutralizing Trachea Animal Infected antibody titer at Nasopharynx PeakRhinorrhea with 10⁴ pfu of time of Chimpanzee Duration Peak TiterDuration Titer Scores indicated virus immunization Number (days)(log₁₀pfu/ml) (days) (log₁₀pfu/ml) Peak Mean cpts-248/404 <10 17 0 <0.70 <0.7 0 0 <10 20 9 <0.7 0 <0.7 0 0 <10 19 8 1.9 0 <0.7 2 0.3 <10 20 92.0 0 <0.7 1 0.2 (mean 4.3) (mean 1.3) (mean 0) (mean <0.7) (mean 0.8)(mean 0.1) 142 21 0 <0.7 0 <0.7 2 0.6 156 22 0 <0.7 0 <0.7 1 0.1 (mean0) (mean <0.7) (mean 0) (mean <0.7) (mean 1.5) (mean 0.4) cpts-530/1009<10 1 9 3.1 0 <1.0 1 0.3 <10 2 10 4.0 10  1.8 1 1.1 <10 3 9 4.0 0 <1.0 10.6 <10 4 9 3.3 0 <1.0 1 0.5 (mean 9.3) (mean 3.6) (mean 2.5) (mean 1.2)(mean 1.0) (mean 0.6) 259 23 8 3.0 0 <0.7 1 0.1 190 24 7 1.2 0 <0.7 10.2 (mean 7.5) (mean 2.1) (mean 0) (mean <0.7) (mean 1.0) (mean 0.2)cpts-248 <10 25 10 4.6 8 5.4 1 0.2 <10 26 10 4.5 6 2.2 1 0.1 <10 27 94.7 10  2.1 1 0.1 <10 28 9 4.2 8 2.2 1 0.1 (mean 9.5) (mean 4.5) (mean8.0) (mean 3.0) (mean 1.0) (mean 0.1) 290 29 13 4.2 0 <0.7 2 0.4 213 3016 4.7 0 <0.7 3 0.9 (mean 14.5) (mean 4.5) (mean 0) (mean <0.7) (mean2.5) (mean 0.7)

TABLE 17 Serum antibody response of chimpanzees immunized on day 0 withRSV cpts-248/404, cpts-248, or cpts-530/1009, in the presence or absenceof passively-transferred antibodies, and challenged 4 to 6 weeks laterwith wild-type RSV A2. RSV F RSV G Day 0 Day 0 Animals (48 hrs. (48 hrs.infected after after with Infused Prior infusion 28 days Prior infusion28 days indicated No. with to of post- to of post- virus of animalsantibodies study antibodies) Postimmunization¹ challenge² studyantibodies) Postimmunization¹ challenge² cpts-248/404 4 no <40 <40 6,4002,560 60 60 1,000 1,600 2 yes <40 1,600 640 25,600 100 100 1,600 21,760cpts-530/1009 4 no <40 <40 6,400 10,240 <40 <40 10,240 2,560 2 yes <401,600 640 10,240 40 100 400 10,240 cpts-248 4 no <40 <40 7,840 6,400 <40<40 250 2,560 2 yes <40 640 1,600 5,400 40 40 1,600 5,440 Neutralizing³Day 0 Animals (48 hrs. infected after with Prior infusion 28 daysindicated to of post virus study antibodies) Postimmunization¹challenge² F G cpts-248/404 <10 <10 208 382 0.2 0.1 <10 199 111 92,6814.3 3.6 cpts-530/1009 <10 <10 256 2,521 1.0 0.3 <10 225 52 37,641 3.73.7 cpts-248 <10 <10 147 338 0.1 0.1 <10 252 119 26,616 4.9 4.9¹The day on which postimmunization titer was determined was also the dayon which challenge was performed, i.e., day 28 for animals not infusedwith antibody, day 42 for animals infused.²Values determined from samples taken 28 days after challenge. Challengeperformed on day 28 postimmunization for animals not infused withantibody, day 42 for animals infused.³Determined by complement-enhanced 60% plaque reduction of RSV A2 inHEp-2 cell monolayer cultures. Neutralizing antibody titer representsthe mean value from two tests.

TABLE 18 Immunization of chimpanzees with RSV cpts-248, cpts-248/404, orcpts-530/1009 cpts-530/1009 induces resistance to wild-type RSV A2challenge 4-6 weeks later. Passively- Replication of RSV A2 challengevirus^(a) transferred Trachea RSV Nasopharynx Peak Rhinorrhea Virus usedfor antibodies Chimpanzee Duration Peak Titer Duration Titer Scoresimmunization present Number (days) (log₁₀pfu/ml) (days) (log₁₀pfu/ml)Mean^(b) Peak cpts-248/404 no  17^(c) 0 <0.7 0 <0.7 0 0 no  18^(c) 8 3.40 <0.7 0 0 yes 21 6 2.7 0 <0.7 0.5 2 yes 22 0 <0.7 0 <0.7 0 0cpts-530/1009 no  1 7 2.1 0 <0.7 0 0 no  2 0 <0.7 0 <0.7 0 0 yes 23 62.5 0 <0.7 0.5 1 yes 24 7 2.0 0 <0.7 0.2 1 cpts-248 no  25^(c) 5 2.7 0<0.7 0 0 no  26^(c) 9 1.8 0 <0.7 0 0 yes 29 0 <0.7 0 <0.7 0 0 yes 30 62.4 0 <0.7 1.2 3 none no  13^(d) 9 5.1 13 5.4 1.0 1 no  14^(d) 9 6.0 86.0 1.7 4 no  15^(c) 13 5.3 8 5.9 2.1 3 no  16^(c) 9 5.4 8 5.6 1.0 3^(a)Animals which were immunized with indicated virus 4 to 6 weeks priorwere challenged with 10 pfu of RSV A2 wild-type virus.^(b)Mean rhinorrhea scores represent the sum of scores during the eightdays of peak virus shedding divided by eight.^(c)Animals from Crowe, et al., Vaccine 12: 691-699 (1994).^(d)Animals from Collins, et al., Vaccine 8: 164-168 (1990).

EXAMPLE II Use of Cold Adaptation to Attenuate cpRSV Mutants

This Example describes the introduction of growth restriction mutationsinto incompletely attenuated host range-restricted cpRSV strains byfurther passage of the strains at increasingly reduced temperatures toproduce derivative strains which are more satisfactorily attenuated foruse in human vaccines.

These cold-adaptation (ca) approaches were used to introduce furtherattenuation into the cpRSV 3131 virus, which is incompletely attenuatedin seronegative children.

Under the first strategy, a parent stock of cold-passaged RSV A2 (cpRSV3131) obtained from Flow Laboratories was prepared by passage in MRC-5cells at 25° C. as described in Example 1. Briefly, cold-passaged viruswas inoculated into MRC-5 or Vero cell monolayer culture at amultiplicity of infection of ≧0.01 and the infected cells were incubatedfor 3 to 14 days before subsequent passage. Virus was passaged over 20times at 20-22° C. to derive more attenuated virus. The technique ofrapid passage, as soon as the first evidence of virus replication isevident (i.e., 3 to 5 days), was preferable for selection of mutantsable to replicate efficiently at low temperatures. Additionally, an RSVsubgroup B strain, St. Louis/14617/85 clone 1A1, was isolated in primaryAfrican Green monkey kidney cells, passaged and cloned in MRC cells(1A1-MRC14), and cold-passaged 52 times in MRC-5 or Vero cells at 32 to22° C.

A second strategy employed a biologically cloned derivative of theuncloned parental cpRSV 3131 virus. This virus was biologically clonedin bovine embryonic kidney (BEK) cells [the tissue used to originallyderive the cpRSV 3131 virus—see Friedewald et al., J. Amer. Med. Assoc.204:690-694 (1968)]. This cloned virus was then passaged at 10 dayintervals in Vero cells at low temperature. Alternatively, the cpRSV3131 virus was cloned by two serial terminal dilutions (TD2P4) in MRC-5cells and passaged at 10-day intervals in MRC-5 or Vero cells.

The third strategy involved selection of mutants that produce largeplaques at low temperature. An RSV 3131 derivative virus designatedplaque D1 that produces large plaques at 25° C. has been identified.This virus was derived from the third passage (P3) level of the cp3131-1(BEK) lineage cp3131-17 (BEK) lineage. The largest plaque produced by P3virus was amplified at 32° C., then re-plaqued at 25° C. Once again thelargest plaque was selected, amplified, and re-plaqued. After five suchcycles, large placque mutant virus D1 was obtained. D1 was biologicallycloned by two additional cycles of plaque-to-plaque purification at 25°C.

Biologically cloned virus D1 produces distinctly and uniformly largerplaques at 25° C. than cp3131 or wild type virus A2. Thus D1 is coldadapted by the criterion of large plaque size at 25° C. Efficiency ofplaque formation studies demonstrated that D1 is not temperaturesensitive. At 37° C., D1 plaques are indistinguishable from those ofwild-type RSV or cp3131, suggesting that D1 is not restricted in growthat this temperature. Consistent with this, D1 produces extensivecytopathic effects in Vero cell monolayers at 37° C. and 40° C. (i.e.the highest temperatures tested).

EXAMPLE III Introduction of Further Attenuating Mutations into ts-RSV

This Example describes the use of ts mutants as parental viruses toproduce more completely attenuated strains. Two RSV A2 ts mutants wereselected for this process, namely ts-4 and ts-1 NG1. Two distinctmethods were chosen to introduce additional mutations into the RSV tsmutants. First, the incompletely attenuated RSV ts mutant was subjectedto chemical mutagenesis, and mutagenized progeny that are moretemperature-sensitive with regard to plaque formation were selected forfurther analysis. Second, the RSV ts mutants were passaged at lowtemperature to select RSV nts mutants with the ca phenotype, i.e.,increased capacity to replicate at suboptimal temperature compared towild-type parental virus.

A parent stock of ts-1 NG1 virus was prepared from Flow Laboratories LotM4 of live Respiratory Syncytial Virus (A-2) ts-1 NG-1 mutant, MRC-5grown virus. This mutant, derived from the ts-1 mutant by a second roundof mutagenesis using nitrosoguanidine, possesses two or more independentts mutations, but still induces substantial rhinorrhea in susceptiblechimpanzees. This virus was passaged twice in Vero cells at 32° C. tocreate a ts-1 NG-1 suspension for mutagenesis. The virus was then grownin the presence of 4×lO⁻⁴M 5-fluorouracil to induce additional mutationsduring replication or was exposed to 5-azacytidine at 36° C. after5-fluorouracil treatment. The mutagenized stock was then analyzed byplaque assay on Vero cells that were maintained under an agar overlay,and, after an appropriate interval of incubation, plaques wereidentified microscopically. 586 plaques were picked, and the progeny ofeach plaque were separately amplified by growth on fresh monolayers ofVero cells. The contents of each of the tissue cultures inoculated withthe progeny of a single plaque of mutagenized ts-1 NG-1 virus wereseparately harvested when cytopathic effects on the Vero cells appearedmaximal. Progeny virus that was more temperature-sensitive than ts-1 NG1was sought by titering these plaque pools on HEp2 cells at 32° C. and36° C. Any virus exhibiting greater temperature sensitivity than ts-1NG1 (i.e., 100-fold or greater reduction in titer at restrictivetemperature [36° C.] compared to 32° C.) was evaluated further. Sixplaque progeny more ts than the tsRSV ts-1 NG-1 parent virus wereidentified and these strains were biologically cloned by serialplaque-purification on Vero cells three times, then amplified on Verocells. The cloned strains were titered at 32° C., 35° C., 36° C., 37°C., and 38° C. (efficiency of plaque formation assay) to confirm theirts phenotype. Efficiency of plaque formation data generated by assay onHEp-2 cells further confirmed the phenotype of the six mutants (Table19).

The two most ts viruses, A-20-4 and A-37-8, were highly attenuated inmice compared to their ts-1 NG1 parent virus, indicating thatacquisition of increased level of temperature sensitivity wasaccompanied by augmented attenuation (Table 20). These viruses wereinfectious for mice because they induced an antibody response. The ts-1NG1/A-20-4 virus is attenuated for chimpanzees (Table 21) and infectionof chimpanzees with ts-1 NG1/A-20-4 induced resistance to wild-typevirus challenge (Table 22). Significantly, rhinorrhea does not occur.

Mutagenesis of the ts-4 virus was also performed, using the same methodas for mutagenesis of ts-1 NG1, virus. Mutations were also introducedinto the ts-4 viruses by cold-passage. The ts-4 virus replicates to hightiter at 22° C. after 43 cold-passages. Six plaque progeny that weremore ts than the RSV ts-4 parent virus were identified (Table 23). Thets-4 cp-43 is even further restricted in replication in Balb/c mice(Table 24). TABLE 19 Efficacy of plaque formation of ts-1NG1 derivativesTiter (log₁₀pfu/ml) at indicated temperature Virus 32° 35° 36° 37° 38°A-204 (4-1)^(a) 5.9* <1 <1 <1 <1 A-37-8 (1-2)^(a) 6.3 6.3 <1 <1 <1A-15-7 3.5 ND 2.1 1.5 <1 A-25-8 5.3 ND 5.0* 4.8* <1 A-21 5.1** ND 4.8**4.5** <1 Ts1NG1 6.6 6.6 6.5 6.6 <1^(a)3x plaque purified*Small-plaque phenotype (<50% wild-type plaque size)**Pinpoint-plaque phenotype (<10% wild-type plaque size)ND = Not Done

TABLE 20 Replication of ts-1 NG1 parent and progency viruses in Balb/cmice Dose Day Post- Titer in Lung Titer in Lung Virus (log₁₀pfu)Infection 32° 38° 32° 38° A2 wt 6.1 4  4.66 ± 0.32^(a) 4.80 ± 0.16 3.18± 4.0  3.29 ± 0.33 5 5.18 ± 0.33 5.25 ± 0.23 3.40 ± 2.0  3.47 ± 0.17Ts1NG1 5.8 4 4.31 ± 0.17 <2.0 2.82 ± 0.25 <2.0 5 3.98 ± 0.12 <2.0 2.74 ±0.31 <2.0 Ts1NG1/A-20-4 6.1 4 <2.0 <2.0 <2.0 <2.0 5 <2.0 <2.0 <2.0 <2.0Ts1NG1/A-37-8 6.3 4 <2.0 <2.0 <2.0 <2.0 5 <2.0 <2.0 <2.0 <2.0^(a)Mean log₁₀ pfu/g of indicated tissue ± standard error. 6animals/group.

TABLE 21 Replication of of ts-1 NG1/A-20-4, ts-1 NG1, ts-1 or wild-typeRSV A2 in the upper and lower respiratory tract of seronegativechimpanzees Virus Replication Animal infected Nasopharynx Trachea withindicated Route of Chimpanzee Duration^(b) Peak titer Duration^(b) Peaktiter Rhinorrhea Scores virus Inoculation^(a) number (Days)(log₁₀pfu/ml) (Days) (log₁₀pfu/ml) Mean^(c) Peak ts-1NG1/A-20-4 IN + IT15  0 <0.7  0 <0.7 0 0 IN + IT 16  0 <0.7  0 <0.7 0 0 IN + IT 17  0 <0.7 0 <0.7 0 0 IN + IT 18 16^(d) 2.7  0 <0.7 0 0 mean 4.0 mean 1.2 mean 0mean <0.7 mean 0 mean 0 ts-1 NG1 IN 19^(e)  8 4.2  0 <1.1 0.6 1 IN20^(e)  7 3.9  0 <1.1 0.7 1 IN 21^(e) 13 5.4  0 <1.1 0.4 1 IN 22^(e) 105.2 10d 3.7^(d) 0.6 2 mean 9.5 mean 4.7 mean 2.5 mean 1.8 mean 0.6 mean1.3 ts-1 IN 23^(e) 16 3.4  0 <1.1 0.4 1 IN 24^(e) 13 4.4  0 <1.1 1.0 3IN 25^(e) 13 5.0 13d 2.2 2.0 4 IN 26^(e) 10 3.4  0 <1.1 1.0 2 mean 13mean 4.1 mean 3.3 mean 1.4 mean 1.1 mean 2.5 A2 wild-type IN  9^(b)  95.1 13 5.4 1.0 1 IN 10^(b)  9 6.0  8 6.0 1.7 4 IN + IT 11^(e) 13 5.3  85.9 2.1 3 IN + IT 12^(e)  9 5.4  8 5.6 1.0 3 mean 10 mean 5.5 mean 9.3mean 5.7 mean 1.4 mean 2.8^(a)IN = Intranasal only; IN + IT = Both intranasal and intratrachealadministration.^(b)Indicates last day post-infection on which virus was recovered.^(c)Mean rhinorrhea score represents the sum of daily scores for aperiod of eight days surrounding the peak day of virus shedding, dividedby eight. Four is the highest score; zero is the lowest score.^(d)Virus isolated only on day indicated.^(e)Animals from Crowe, et al., Vaccine 11: 1395-1404 (1993).

TABLE 22 Immunization of chimpanzees with 10⁴ pfu of RSV ts-1NG1/A-20-4, ts-1 NG1, or ts-1 induces resistance to 10⁴ pfu RSV A2wild-type virus challenge on day 28. Serum neutralizing antibody titerVirus Recovery (reciprocal log₂) on Nasopharynx Trachea Rhinorrhea dayindicated^(b)) Virus used to Chipanzee Duration Peak titer Duration Peaktiter scores Day 49 or immunize animal number (Days) (log₁₀pfu/ml)(Days) (log₁₀pfu/ml) Mean^(a) Peak Day 28 56 ts-1 NG1/A-20-4 15 0 <0.7 0<0.7 0 0 <3.3 10.7 16 0 <0.7 0 <0.7 0 0 <3.3 11.9 17 0 <0.7 0 <0.7 0 05.3 10.3 18 3 2.0 0 <0.7 0 0 8.2 11.8 mean 0.8 mean 1.0 mean 0 mean <0.7mean 0 mean 0 mean 5.0 mean 11.2 ts-1 NG1 19^(b) 0 <0.7 0 <1.1 0 0 11.19.8 20^(b) 0 <0.7 0 <1.1 0 0 12.7 9.1 21^(b) 0 <0.7 0 <1.1 0 0 10.8 11.022^(b) 0 <0.7 0 <1.1 0 0 10.0 8.6 mean 0 mean <0.7 mean 0 mean <1.1 mean0 mean 0 mean 11.1 mean 9.6 ts-1 23^(b) 0 <0.7 0 <1.1 0 0 9.4 10.529^(b) 0 <0.7 0 <1.1 0 0 12.4 12.8 25^(b) 5 0.7 0 <1.1 0 0 9.0 9.626^(b) 5 0.7 0 <1.1 0 0 13.4 12.0 mean 2.5 mean 0.7 mean 0 mean <1.1mean 0 mean 0 mean 11.0 mean 11.2 none  9^(c) 9 5.1 13 5.4 1.0 1 <3.311.0 10^(c) 9 6.0 8 6.0 1.7 4 <3.3 9.8 11^(b) 13 5.3 8 5.9 2.1 3 <3.39.4 12^(b) 9 5.4 8 5.6 1.0 3 <3.3 14.5 mean 10 mean 5.5 mean 9.3 mean5.7 mean 1.5 mean 2.8 mean <3.3 mean 11.1^(a)Mean rhinorrhea score represents the sum of scores during the eightdays of peak virus shedding divided by eight. Four is the highest score;zero is the lowest score.^(b)Animals from Crowe, et al., Vaccine 11: 1395-1404 (1993).^(c)Animals from Collins, et al., Vaccine 8: 164-168 (1990).^(d)Serum neutralizing titers in this table were determined in a newassay simultaneously with other specimens represented in the table.

TABLE 23 The efficiency of plaque formation of six mutants derived fromRSV ts-4 and tested in HEp-2 cells at permissive and restrictivetemperatures, compared with controls. The titer of virus (log₁₀pfu/ml)Small- Shut-off at the indicated temperature (° C.) plaques temperatureVirus 32 33 34 35 36 37 38 39 40 at 32° C. (° C.)¹ A2 wild-type 5.7 5.85.5 5.5 5.3 5.5 5.5 5.4 5.5 no >40 ts-4 4.5 4.7 4.4 4.7 4.7 4.1 3.7 3.02.5 no 40 ts-4 cp-43 6.2 6.1 6.1 6.0 4.4* 4.2** 1.7** <0.7** <0.7 no 37ts-4/20.7.1 6.0 5.9 5.7 5.7* 4.5** 1.8 <0.7 <0.7 <0.7 no 37 ts-4/19.1.25.8 5.7 5.5 5.6* 4.4** <0.7 <0.7 <0.7 <0.7 no 37 ts-4/15.8.2 5.3* 5.4*4.8* 4.9* 2.8** <0.7 <0.7 <0.7 <0.7 yes 36 ts-4/29.7.4 5.7 5.6 5.6 5.7*<0.7 <0.7 <0.7 <0.7 <0.7 no 36 ts-4/31.2.4 4.7 4.2 4.1 4.0* <0.7 <0.7<0.7 <0.7 <0.7 no 36¹Shut-off temperature is defined as the lowest restrictive temperatureat which a 100-fold or greater reduction of plaque titer is observed(bold figures in table).*Small-plaque phenotype (<50% wild-type plaque size)**Pinpoint-plaque phenotype (<10% wild-type plaque size)

TABLE 24 Replication of RSV ts-4 and RSV ts-4 cp-43 in Balb/c mice¹Shutoff Virus titer (mean log₁₀pfu/g tissue Virus used to temperature ofof six animals ± standard error) infect animals: virus (° C.) Nasalturbinates Lungs A2 wild-type >40 5.0 ± 0.14 5.2 ± 0.05 ts-4 39 4.3 ±0.09 4.7 ± 0.11 ts-4 cp-43 37 2.1 ± 0.09 2.7 ± 0.27¹Mice were administered 10^(6.3)p.f.u. intranasally under lightanesthesia on day 0, then sacrificed by CO₂ asphyxiation on day 4.

EXAMPLE IV RSV Subgroup B Vaccine Candidates

This Example describes the development of RSV subgroup B virus vaccinecandidates. The same approach used for the development of the subgroup Amutants of the invention was utilized for the subgroup B viruses. Aparent stock of wild-type B-1 RS virus was cold-passaged 52 times inVero cells at low temperature (20-25° C.) and the virus was subjected toplaque purification at passages 19 and 52. Three of the clones derivedfrom the passage 52 suspension were evaluated independently, and oneclone, designated RSV B-1cp52/2B5, was selected for further evaluationbecause it was highly attenuated in the upper and lower respiratorytract of the cotton rat (Table 25). An evaluation of several clones atdifferent passage levels of the cp RSV B-1 virus indicate that the RSVB-1cp52/2B5 mutant sustained multiple mutations that independentlycontribute to its attenuation phenotype. The RSV B-1cp52/2B5 mutantretained its attenuation phenotype following prolonged replication inimmunosuppressed cotton rats (Table 26). This finding of a high level ofgenetic stability is consistent with the fact that it possesses threemutations contributing to the attenuation phenotype.

Further evaluation of the subgroup B mutants in order to characterizethem in a similar manner as the subgroup A mutants, was carried out inCaribbean Green monkeys (Tables 27 and 28) and chimpanzees (Table 29).Monkeys immunized with either RSV B-1 cp-23 or cp52/2B5 were resistantto replication of RSV B-1 wild-type virus, indicating that infectionwith the highly attenuated derivatives of the RSV B-1 wild-type viruswas sufficient to induce resistance to wild-type challenge (Table 27).

The results in the seronegative chimpanzee, like that in the Greenmonkeys, clearly evidence the attenuation of the RSV B-1cp52/2B5 in theupper and lower respiratory tracts.

The RSV B-152/2B5 mutant has been further mutagenized with5-fluorouracil and the resulting plaques picked and screened at 32° vs.38° C. for the ts phenotype. The selected cpts mutants wereplaque-purified three times in Vero cells and then amplified twice inVero cells. As a result, seven cpts mutants of RSV B-1cp52/2B5 have beenidentified (Table 30) and their level of replication in cotton rats hasbeen studied (Table 31). One of these mutants, namely cpts176, wasfurther mutagenized and a series of mutant derivatives were obtainedthat were more ts in vitro than the RSV B-1cpts 176 parent virus (Table32).

As with the subgroup A mutants of the invention, the subgroup B mutantsare infectious and exhibit a significant degree of attenuation forcotton rats, monkeys, and chimpanzees. Despite attenuation in vivo, theRSV B-1 cp mutant viruses induced resistance in monkeys againstwild-type challenge. The ts mutants of the RSV B-1 cpts52/2B5 virus areattenuated and demonstrate a more restricted level of replication in thenasopharynx and lungs of the cotton rat than the RSV B-1 52/2B5 parentvirus. TABLE 25 Replication in cotton rats of RSV B-1 wild-type comparedwith five plaque-purified cold-passaged mutants derived from RSV B-1, intwo separate experiments. Virus recovery (log₁₀pfu/g tissue) on day 4*Virus used to infect Nasal turbinates Lungs animals on day 0** Exp. 1Exp. 2 Exp. 1 Exp. 2 RSV B-1 wild-type 4.7 ± 0.14 5.1 ± 0.10 5.4 ± 0.155.8 ± 0.08 RSV B-1 cp-12/B1A nd 3.3 ± 0.15 nd 4.4 ± 0.10 RSV B-1 cp-23nd 2.4 ± 0.36 nd 3.2 ± 0.31 RSV B-1 cpsp-52/1A1 1.7 ± 0.11 2.1 ± 0.273.0 ± 0.13 2.3 ± 0.07 RSV B-1 cp-52/2B5 1.8 ± 0.25 2.2 ± 0.3  1.8 ± 0.111.5 RSV B-1 cp-52/3C1 1.8 ± 0.14 nd 1.8 ± 0.14 nd RSV A2 5.9 ± 0.09 5.4± 0.07 6.6 ± 0.06 6.1 ± 0.06 RSV A2 cpts530/1009 3.2 ± 0.11 2.1 ± 0.222.1 ± 0.19 1.7 ± 0.12*Virus recovery determined by titration of tissue homogenates on Verocell monolayer cultures at 32° C. with a 10-day incubation in Experiment1, 7-day incubation in Experiment 2.**Cotton rats infected intranasally with 10^(5.5) pfu of indicatedvirus.nd = not done

TABLE 26 Growth in cotton rats of day 14 isolates* from RSV B-1 cp52/2B5infected immunosuppressed cotton rats compared with controls VirusRecovery Virus titer on day 4 in indicated tissue RSV B-1 wild-typeVirus (mean log₁₀pfu/g tissue ± (log₁₀pfu/g) infected standard error ofthe mean) Nasal animals^(a) Nasal turbinates^(b) Lungs^(c) turbinatesLungs RSV B-1 3.9 ± 0.03 (6/6) 4.8 ± 0.12 (6/6) — — wild-type RSV B-12.0 ± 0.07 (8/8) <1.5 (0/8) 1.9 >3.3 cp 52/2B5 isolate 1 1.5 ± 0.13(5/8) 1.5 ± 0.04 (1/8) 2.5 3.3 isolate 2 1.5 ± 0.13 (6/8) <1.5 (0/8)2.4 >3.3 isolate 3 1.5 ± 0.16 (3/8) <1.5 (0/8) 2.5 >3.3 isolate 4 1.3 ±0.09 (4/8) <1.5 (0/8) 2.6 >3.3 isolate 5 1.2 ± 0.00 (2/8) <1.5 (0/8)2.7 >3.3 isolate 6 1.2 ± 0.00 (3/8) <1.5 (0/8) 2.7 >3.3 isolate 7 1.3 ±0.06 (3/8) <1.5 (0/8) 2.7 >3.3*Isolates were virus suspensions obtained following amplification by oneVero cell tissue culture passage of virus present in the original nasalturbinate homogenate on day 14 of an immunosuppressed cotton rat.^(a)Groups of 8 cotton rats infected with 10^(5.5) pfu of indicatedvirus in a 0.1 ml inoculum on day 0.^(b)( )indicates the numbers of animals from which virus was detected at1.2 log₁₀pfu/g or greater.^(c)( )indicates the numbers of animals from which virus was detected at1.5 log₁₀pfu/g or greater.

TABLE 27 Table 27. Replication in Caribbean Green monkeys of RSV A2 andRSV B-1 wild-types compared with that of two cold-passaged mutantsderived from RSV B-1, followed by homologous or heterologous RSV A2 orB-1 wild-type challenge Challenge Virus used Immunization NP Tracheal toinfect NP swab Tracheal Lavage Chal- swab Lavage animals Peak Days PeakDays lenge Peak Peak on day 0^(a) titer^(b) shed^(b) titer shed virustiter^(b) titer^(b) A2 3.4 9 <0.7 0 A2 <0.7 <0.7 3.5 7 <0.7 0 A2 <0.7<0.7 3.5 9 4.8 10  A2 <0.7 <0.7 3.2 8 0.7 7 A2 <0.7 <0.7 1.7 6 <0.7 0B-1 <0.7 <0.7 3.5 10  <0.7 0 B-1 <0.7 <0.7 2.4 8 0.7 0 B-1 <0.7 <0.7 4.29 <0.7 0 B-1 <0.7 <0.7 mean mean mean 3.2   8.3 1.2 B-1 2.8 9 1.5 10*B-1 <0.7 <0.7 2.3 9 1.9 7 B-1 <0.7 <0.7 2.2 7 1.7 10* B-1 <0.7 <0.7 2.29 1.3 10* B-1 <0.7 <0.7 1.6  8* 1.2  5* A2 <0.7 <0.7 2.1 10  1.7  7* A2<0.7 <0.7 mean mean mean mean mean 2.2   8.7 1.6 <0.7 <0.7 B-1 1.8 14 <0.7 0 B-1 <0.7 <0.7 cp-23 1.3 5 <0.7 0 B-1 <0.7 <0.7 2.0 8 0.7 10  B-1<0.7 <0.7 1.7 4 <0.7 0 B-1 <0.7 <0.7 mean mean mean mean mean 1.7   7.8<0.7 <0.7 <0.7 B-1 1.3 8 <0.7 0 B-1 <0.7 <0.7 cp-52 1.3 4 <0.7 0 B-1<0.7 <0.7 1.3 7 <0.7 0 B-1 <0.7 <0.7 0.7  3* <0.7 0 B-1 <0.7 <0.7 meanmean mean mean mean 1.2   5.5 <0.7 <0.7 <0.7^(a)Animals infected intratracheally and intranasally with 10^(5.5)p.f.u. virus at each site in a 1.0 ml inoculum on day 0.^(b)Log₁₀pfu/ml titers determined by plaque assay on HEp-2 cellmonolayer cultures for RSV A2, and Vero cell monolayer cultures for RSVB-1 and its derivatives.*Virus detected only on day indicated.

TABLE 28 Neutralizing antibody response of Caribbean Green Monkeysinfected with RSV A2, RSV B-1, or B-1 cp derivatives, then challengedwith homologous or heterologous wild-type virus one month later. 60%Plaque reduction serum neutralizing titer against Animals infectedindicated virus (reciprocal mean) on day 0 with Day 28 RSV A2 RSV B-1indicated virus challenge virus Post-infection Post-challengePost-infection Post-challenge (number of animals) (number of animals)Day 0 (day 28) (day 56) Day 0 (day 28) (day 56) A2 (8) A2 (4) <10 53,23240,342 <10 1,552 1,911 B-1 (4) 23,170 1,911 B-1 (6) B-1 (4) <10 3,3273,822 <10 2,048 2,521 A2 (2) 30,574 35,120 B-1 cp23 (4) B-1 (4) <106,208 10,086 <10 4,705 7,132 B-1 cp-52/2B5 (4) B-1 (4) <10 194 16,384<10 239 3,822

TABLE 29 The replication of RSV B-1 or RSV B-1 cp-52 in seronegativechimpanzees following simultaneous intratracheal and intranasaladministration.^(a) Virus replication Nasopharynx Trachea Animalinfected with Infection Duration^(b) Peak titer Duration^(b) Peak titerRhinorrhea score indicated virus on day 0 dose (pfu) Exp. (days)(log₁₀pfu/ml) (days) (log₁₀pfu/ml) Peak Mean^(c) RSV B-1 wild-type 10⁴ 19 3.7 8 3.2 1 0.5 1 10  3.5 0 <0.7 2 0.9 1 10  2.8 0 <0.7 3 1.1 1 10 2.7 8 3.4 3 0.9 avg. 9.8 mean 3.2 avg. 4.0 mean 2.0 mean 2.3 mean 0.910⁵ 2 7 2.8 8 1.0 1 1.0 2 7 3.3 4 3.9 3 1.3 avg. 7.5 mean 3.1 avg. 6.0mean 2.5 mean 2.0 mean 1.1 B-1 cp-52/2B5 10⁴ 1 5 1.5 0 <0.7 0 0 1 0 <0.70 <0.7 0 0 10⁵ 3 0 <0.7 0 <0.7 0 0 3 0 <0.7 0 <0.7 0 0 avg. 1.2 mean 0.9mean 0 mean <0.7 mean 0 mean 0^(a)These data were combined from three separate experiments, theinfection dose of indicated virus in the first experiment was 10⁴, thesecond and third experiments were 10⁵.^(b)Indicates the last day post-infection on which virus was recovered.^(c)Mean rhinorrhea score represents the sum of daily scores for aperiod of eight days surrounding the peak day of virus shedding, dividedby eight. Four is the highest score; zero is the lowest score.

TABLE 30 The efficiency of plaque formation of eight mutants derivedfrom RSV B-1 cp52/2B5 Plaque titer (log₁₀pfu/ml in Vero of HEp-2 Cellsat indicated temperatures (° C.) HEp-2 Shutoff Vero HEp-2 temp RVS 32 3235 36 37 38 39 (° C.) B-1 6.1 5.8 5.7 5.6 5.6 5.7 5.5 >39 wild-type B-15.9 5.4 5.2 5.1 5.0 5.0 4.7** >39 cp52/2B5 cpts452 6.1 5.6 5.2 5.2 3.3**3.1** <0.7 37 cpts1229 5.7 5.1 4.9 5.1 4.4** <0.7 <0.7 38 cpts1091 5.75.1* 4.7** 5.2** <0.7 <0.7 <0.7 37 cpts784 5.1 4.3* 4.0** 4.1** <0.7<0.7 <0.7 37 cpts176 6.1 5.4* 4.8* 5.0** <0.7 <0.7 <0.7 37cptssp1415^(a) 5.8 <0.7 <0.7 <0.7 <0.7 <0.7 <0.7 38 cpts1324 5.9 5.15.0* 5.0 <0.7 <0.7 <0.7 37 cpts1313 5.7 3.9** 3.0** <0.7 <0.7 <0.7 <0.736 AS 6.4 6.3 6.3 6.3 6.3 6.3 6.3 >39 A2/248 6.3 6.3 6.2 6.3 5.8 <0.7<0.7 38 A2/ 4.4 4.3 3.3 4.0 <0.7 <0.7 <0.7 37 248/404 A2/ 4.8 4.8 4.84.4 <0.7 <0.7 <0.7 37 248/955*Small-plaque phenotype (<50% wild-type plaque size).**Pinpoint-plaque phenotype (<10% wild-type plaque size).^(a)At 32° C., no plaques were observed. Therefore, no shut-offtemperature was determined by efficiency of plaque formation. The mutantwas assigned a shutoff temperature of 38° C. in HEp-2 cell culture asdetermined by a 100-fold decrease in virus yield (TCID₅₀) in liquidmedium overlay.Bold figures denote shutoff temperatures (defined as the lowestrestrictive temperature at which a 100-fold or greater reduction ofplaque titer was observed).

TABLE 31 Level of replication in cotton rats of seven ts mutants derivedfrom RSV B-1 cp-52/2B5 Replication in cotton rats¹ (mean log₁₀pfu/gtissue of six animals ± s.e.) RSV Nasal turbinates Lungs B-1 wild-type4.3 ± 0.05  (6/6)² 4.4 ± 0.25 (6/6) B-1 cp52/2B5 1.7 ± 0.11 (6/6) <1.5(0/6) cpts452 1.4 ± 0.1  (3/6) <1.5 (0/6) cpts1091 1.7 ± 0.07 (4/6) <1.5(0/6) cpts784   1.5 (1/6) <1.5 (0/6) cpts1229 1.4 ± 0.15 (3/6) <1.5(0/6) cpts176 1.5 ± 0.17 (3/6) <1.5 (0/6) cptssp1415 <1.2 (0/6) <1.5(0/6) cpts1324 <1.2 (0/6) <1.5 (0/6)¹Cotton rats were inoculated intranasally with 4.5-5.8 log₁₀pfu underlight anesthesia on day 0, then sacrificed by CO₂ asphyxiation on day 4.²Titer from samples containing virus only. Parentheses indicate fractionof samples containing virus.

TABLE 32 The efficiency of plaque formation of 14 mutants derived fromRSV B-1 cpts176, compared with controls In vitro efficiency of plaqueformation in HEp-2 cell monolayer culture The titer of virus(log₁₀pfu/ml) at the indicated Shut-off temperature (° C.) temperatureRSV 32 35 36 37 (° C.)¹ B-1 wild-type 5.6 5.5 5.4 5.3 >39   B-1 cp52/2B55.7 5.7 5.6 5.3 >39   B-1 cpts176 5.5 3.5 3.0 1.9 36/37 176/645 3.8 3.02.6** <0.7 37 176/860 3.1 2.5 2.4 <0.7 37 176/196 3.3 2.5 2.0** <0.7 37176/219 2.6 2.3 2.0** <0.7 37 176/18 4.0 3.2 <0.7 <0.7 36 176/73 2.6 2.0<0.7 <0.7 36 176/1072 3.2 2.3 <0.7 <0.7 36 176/1038 2.8 2.2 <0.7 <0.7 36176/81 2.2 2.0 <0.7 <0.7 36 176/1040 3.2 2.0 <0.7 <0.7 36 176/1045 2.51.9 <0.7 <0.7 36 176/517 3.1 2.0** <0.7 <0.7 36 176/273 2.3 <0.7 <0.7<0.7 35 176/427 3.5 <0.7 <0.7 <0.7 35**Pinpoint-plaque phenotype (<10% wild-type plaque size)¹Shut-off temperature is defined as the lowest restrictive temperatureat which a 100-fold or greater reduction of plaque titer is observed(bold figures in table).

To aid in the evaluation and manipulation of RSV B subgroup vaccinecandidates, the complete nucleotide sequence of the wild-type B-1 virushas been determined [SEQ ID NO:2]. This sequence was compared with thesequence of the attenuated B-1 derivative, cp-52/2B5 (cp-52), describedabove. This sequence analysis revealed a large deletion in cp-52spanning most of the SH and G genes, with no predicted ORF for eithergene. More specifically, most of the region spanning the SH and G genesof the cp-52 virus was deleted, retaining only the SH gene-start signaland a portion of the 3′ (downstream) end of the G gene and its gene-endsignal. The remaining SH:G region could encode a chimeric transcript of˜91 nucleotides with no ORF. Northern blot analysis of cp-52 confirmedthat multiple unique polytranscripts contained SH:G read-through mRNAs,consistent with a deletion mutation spanning the SH:G gene junction.Western blot and immunostain assays confirmed that intact G glycoproteinwas not produced by the cp-52 virus. In addition to the long deletion,cp-52 virus contains seven nucleotide differences (Table 33), five ofwhich are coding changes (one in the F gene and four in the L gene), oneis silent (F gene), and one is in the noncoding G:F intergenic region.Importantly, this RSV mutant remains highly infectious in tissue culturedespite the absence of SH and G proteins. These data identify a novelclass of replication competent deletion mutants which provide foralternative or combinatorial approaches to developing recombinant RSVvaccine candidates. TABLE 33 Sequence comparison of RSV B1 and cp-52Genomic Nucleotide* Amino acid change (#) Gene position B1 cp-52†B1→cp-52 G/F 5626 C A** non-coding intergenic F 6318 A G Glu→Gly (218)6460 U C** silent (265) L 10973 G A Arg→Lys (822) 13492 A C Asn→His(1662) 14164 U A** Leu→Ile (1886) 14596 U C** Phe→Leu (2030)*Positive (+) sense.† Nucleotide position 4249-5540 spanning the SH and G genes is deletedin cp-52.**Mutations present in cp-23

Other subgroup B mutants isolated at different passage levels in thecp-52 passage history incorporate various of the cp-52 mutations,depending on passage level (Table 34). Exemplary subgroup B mutants inthis context include RSV B-1 cp-12, RSV B-1 cp-23, RSV B-1 cp-32, RSVB-1 cp-42. Table 34 depicts (as negative sense) the distribution ofthese specific mutations among exemplary B subgroup mutants. This varieddistribution of mutations allows for more refined characterization ofthe attenuating effects of these mutations in the designated strains.For example, cp-23 incorporates the mutations at nucleotide positions5626, 6460, 14164 and 14596 found in cp-52 (Table 34), but has nodifferences from the parental B-1 wild-type strain in the SH and G generegion that is deleted in cp-52. cp-42 incorporates the same SH and Gdeletion as cp-52, while cp32 presents a distinct deletion of sequenceswithin the SH and G genes. TABLE 34 Sequence comparison between RSVB1,RSVB1cp12, cp 23, cp32, cp 42, and cp 52 Nucleotide Changes genome (−)sense Nucl. RSVB1 RSVB1 RSVB1 RSVB1 RSVB1 Amino Acid Gene Pos. RSVB1CP12 ••CP23 CP32 CP42 •CP52 Changes G/F 5626 G G T T *ND T non-codinglGr F 6318 T ND T T ND C Glu→Gly (218) 6460 A A G G *ND G silent (265) L10973 C ND C C T T Arg→Lys (822) 13492 T ND T T T G Asn→His (1662) 14164A A T T T T Leu→Ile (1886) 14596 A G G G G G Phe→**Leu (2030)•Nucleotide region (position 4249-5540) spanning the SH and G genes isdeleted in cp52.••No nucleotide differences from B1 parent found in the SH and G generegion which is deleted in cp52.*These nucleotides are most likely the same as in cp23 and cp 52.**A Leucine at amino acid position 2030 in the L polymerase is alsofound in RSV2B.

EXAMPLE V Bivalent RSV Subgroup A and B Vaccine

Studies with subgroup A and B viruses demonstrate that in vitro, nointerference occurs between wild-type A2 and B-1 viruses, nor betweencpts530/1009 and RSV B-1 cp52/2B5 derivatives in Vero cell monolayercultures. The in vivo results of bivalent infection in cotton rats arepresented in Table 34. These results confirm the in vitro results, whichshow no interference between A-2 and B-1 wild-type RSV, and cpts530/1009and RSV B-1 cp52/2B5. It is expected, therefore, that each vaccine viruswill induce homotypic immunity against wild-type virus, since eachcomponent of the bivalent vaccine replicates to a level in the dualinfection comparable to that seen during single infection. Each virusalone is capable of inducing homotypic resistance against RSV wild-typechallenge. TABLE 35 Bivalent infection of cotton rats with RSV A2 andRSV B-1 viruses or mutant derivatives indicates no in vivo interferenceVirus recovery from indicated tissue (log₁₀pfu/g) Nasal turbinates LungsViruses used to RSV A RSV B RSV A RSV B infect animals* titer titertiter titer A2 5.4 ± 0.08 — 5.8 ± 0.07 — B-1 — 4.6 ± 0.03 — 5.4 ± 0.12A2 + B-1 5.2 ± 0.11 3.6 ± 0.07 5.7 ± 0.08 5.0 ± 0.05 A2 cpts530/1009 3.2± 0.09 — 1.9 ± 0.15 — B-1 cp52 — 2.4 ± 0.08 — <1.5 A2 cpts530/1009+ 2.8± 0.13 2.0 ± 0.14 1.8 ± 0.08 <1.5 B1 cp-52*Groups of six animals infected with 10⁵ pfu intranasally on day 0 in a0.1 ml inoculum.

EXAMPLE VI A Single Mutation in Polymerase (L) Gene Elicits ts Phenotype

This Example describes the specific mutations in the polymerase gene (L)that were produced by chemical mutagenesis of incompletely attenuatedhost range-restricted cpRSV to produce ts strains, cpts248 and cpts530,which are more highly attenuated and suitable for use in RSV vaccinepreparations. As described in the Examples above, cpts248 has been foundto be attenuated, immunogenic, and fully protective against wild-typechallenge in seronegative chimpanzees and is more stable geneticallyduring in vivo replication than previously evaluated tsRSV mutants. Asdescribed above, the cpts248 strain was subjected to chemicalmutagenesis to further reduce residual reactogenicity, yielding a seriesof mutants with increased temperature sensitivity or small plaquephenotype, including cpts248/404. In a like manner, cpts530/1009 wasderived from cpts530.

The genetic bases for increased attenuation and ts phenotype of cpts248and cpts530 were determined by comparing the complete genomic sequenceof these viruses with that of the previously determined sequence of thecpRSV parental virus. The complete nucleotide sequence of cpRSV wasdetermined and compared with that of RSV A2 wild-type virus (alaboratory strain which was not part of the direct passage lineage), aswell as with the sequence of its low passaged wild-type parent virus(RSV A2/HEK7). The cpRSV differs from its RSV A2/HEK7 parent virus byfive nucleotide changes, one in the nucleoprotein (N), two in the fusionprotein (F) gene and two in the polymerase (L) gene. The complete 15,222nucleotide sequence and amino acid sequence of cpts248, cpts248/404,cpts530, cpts530/1009, and cpts530/1030 were determined.

The derivation of the RSV mutants cpts248 and cpts530 from their cpRSVparent by random chemical mutagenesis was described in Example 1. Thevirus suspension used for infecting cells for production of virus to beused as a source of purified viral RNA was a clarified tissue culturesupernatant containing virus that had been passaged four times in liquidmedium in Vero cell monolayer culture following biological cloning(i.e., three plaque-to-plaque passages in Vero cell monolayers underagar).

Cell monolayers were infected at a multiplicity of infection (moi) of0.1 with cpts248, cpts248/404, cpts530, cpts530/1009, or cpts530/1030viruses. Total RNA was prepared from infected cell monolayers whenmoderate CPE was observed (average of 4-5 days postinfection). Thesupernatant fluid was removed by aspiration, and infected cellmonolayers were harvested by lysis with guanidinium isothiocynanate,followed by phenol-chloroform extraction. RNA was reverse transcribedusing Superscript II™ reverse transcriptase (Life Technologies) randomhexamer primers, and reaction conditions as described in protocolssupplied by the manufacturer.

The resulting cDNA was separated from primers using TE-100 spin columns(Clontech, Palo Alto, Calif.) and used as template in polymerase chainreactions (PCR) to generate a series of ten overlapping cDNA clonesspanning the entire RSV genome. The oligonucleotide primers for PCR weredesigned from the nucleotide sequence of the prototype A2 virus (Mink etal., Virology 185:615-624 (1991); Stec et al., Virology 183:273-287(1991); Collins et al., Proc. Natl. Acad. Sci. U.S.A. 88:9663-9667(1991); Connors et al., Virology 208:478-484 (1995)), and had beendemonstrated previously to amplify both the RSV A2 wild-type virus andits derivative, the cpRSV parental virus. Uracil-containingoligonucleotide primers were used for cloning RSV sequences into thepAMP1 vector using the CloneAmp uracil DNA glycosylase system (LifeTechnologies). PCR reactions were performed according to themanufacturer's protocols (Perkin-Elmer, Norwalk, Conn.) and carried outfor 34 cycles of each 1 min. at 92.5° C., 1 min. at 55° C., and 3 min.at 72° C. Each fragment was electrophoresed in a 1% agarose/TAE gel,recovered by the Geneclean II System (Bio101, Vista, Calif.), and clonedinto the pAMP1 vector. Two or more separate clones of each fragment weregenerated from separate PCR reactions. For analysis of the 3′ leaderregion, viral RNA (vRNA) was polyadenylated as described in Mink et al.,Virology 185:615-624 (1991) in a 50 μl reaction. Following incubation at37° C. for 10 minutes, the reaction was stopped with 2 μl of 0.5 M EDTA.The polyadenylated RNA product was purified by extraction with phenolchloroform and ethanol precipitation, and then reverse transcribed intocDNA, amplified by PCR, and cloned using a rapid amplification by the 3′RACE system (Life Technologies). Similarly, for analysis of the 5′trailer region, vRNA was reverse transcribed into cDNA, tailed usingterminal deoxynucleotidyl transferase and dCTP, column purified, madedouble-stranded and amplified by PCR, and cloned using a 5′ RACE system(Life Technologies).

Cloned cDNAs for cpts248 were sequenced from double stranded plasmids bythe dideoxynucleotide method using synthetic oligonucleotide primers(either plasmid primers or RSV specific primers), [α³⁵S]dATP andSequenase 2.0 (United States Biochemicals, Cleveland, Ohio). Differencesbetween the observed sequences and those of the previously publishedparental virus cpRSV were confirmed by sequencing two or more clones inboth forward and reverse directions, and by sequencing uncloned PCRproducts.

Nucleotide sequences of the cpts248/404, cpts530, cpts530/1009, andcpts530/1030 were determined using a different technique. Three to tenoverlapping cDNA clones representing the genome of cptsRSV mutant viruswere generated by RT-PCR of total infected-cell RNA or vRNA. Thecomplete nucleotide sequence of each clone was determined by automatedDNA sequence at the NCI Frederick Cancer (Frederick, Md.) using Taq kit(ABI, Foster City, Calif.) on a M13 sonicated random library constructedin phage M13 for each plasmid insert. Both strands were sequenced anddifferences between the cpRSV and the cptsRSV mutant sequences wereconfirmed by manual sequencing (as above) of a second independentlyderived RT-PCR product using Sequenase 2.0 (USB, Cleveland, Ohio). The3′- and 5′-end sequences were determined as described above.

The complete nucleotide sequence of the 15,222-nucleotide RNA genome ofthe cpts248, cpts248/404, cpts530, cpts530/1009, and cpts530/1030strains were determined. Changes relative to the published sequences ofthe cpRSV and RSV A2/HEK7 (wild-type) parental viruses (Conners et al.,Virology 208:478-484 (1995)) were confirmed on independently derivedcDNA clones of these cptsRSV mutants, and were rechecked in the cpRSVvirus by sequencing an additional clone of cpRSV. An ambiguity in thesequence of the cpRSV virus (compared with the A2/HEK7 wild-type virus)was identified. The ambiguity occurred at position 1,938 from the 3′ endof the negative sense genome and consisted of nucleotide heterogeneity(G or A in positive sense) that would be predicted to encode an aminoacid change (Val or Ile) at amino acid position 267 of the 391 aminoacid-nucleocapsid (N) protein. Thus, the cpRSV actually consisted of amixed population of viruses. This accounts for the initial failure todetect the change at position 1938 in Conners et al., supra. A viruswith the A mutation at position 1,938 was the immediate parent of thecpts248 derivative as well as of the cpts530 sister clone. Thus, thecpRSV which was the immediate parent virus of the cpts derivativecontains five amino acid differences from its A2/HEK parent.

A single nucleotide difference between the cpRSV and cpts248 mutants wasfound at nucleotide position 10,989 (an A to T change) (Table 36). Thismutation occurred within the polymerase (L) translation open readingframe, encoding a predicted amino acid change of gln to leu at aminoacid position 831 in the 2,165 amino acid L protein. The cpts248/404mutant possesses two nucleotide differences from its cpts248 parent, oneat nucleotide 12,046 (T to A) in the L gene, and one at nucleotide 7605(T to C) in the transcription start signal sequence of the M2 gene. Thenucleotide substitution in the L gene resulted in an amino acid changefrom asp to glu at position 1183 in the L protein. Thus after twoindependent rounds of mutagenesis of cpRSV, the cpts248/404 virusacquired two nucleotide changes in the L gene (corresponding to twoamino acid substitutions) and one nucleotide change in the transcriptionstart signal of the M2 gene. Compared to its wild type progenitor,A2/HEK7, the cpts248/404 mutant differs in seven amino acids (four in L,two in F, and one in N) and in one nucleotide in the transcription startsignal of the M2 gene. TABLE 36 Nucleotide Sequence Differences amongcp-RSV, cpts-248, cpts- 248/404 mutant viruses Nucleotide and Amino AcidChange Amino A2 wt cpts- Nucleotide¹ Acid Gene (HEK-7) cp-RSV cpts-248248/404 1938 267 N G (val) A (ile) A (ile) A (ile) 3242 P A A A AA² 6313218 F A (glu) C (asp) C (asp) C (asp) 7228 523 F C (thr) T (ile) T (ile)T (ile) 7605 Gene T T T C Start (M2) 9453 319 L G (cys) A (tyr) A (tyr)A (tyr) 10989 831 L A (gln) A (gln) T (leu) T (leu) 12046 1183 L T (asp)T (asp) T (asp) A (glu) 13565 1690 L C (his) T (tyr) T (tyr) T (tyr)¹Positive sense²Increases genome length by 1 nucleotide.

The cpts530 differs from the parental strain cpRSV by the singleadditional nucleotide substitution of C to A at position 10,060 andresults in a phe to leu amino acid change at position 521 of the Lprotein (Table 37). The cpts530/1009 mutant has a single nucleotidesubstitution at nucleotide 12,002 (A to G) resulting in a met to valsubstitution at amino acid 1169 of the L protein. Compared to its wildtype progenitor, A2/HEK7, the cpts530/1009 mutant differs in seven aminoacids (four in L, two in F, and one in N).

The cpts530/1030 mutant has a single additional nucleotide substitutionat nucleotide 12458 (T to A) resulting in a Tyr to Asn substitution atamino acid 1321 of the L protein. Compared to its wt progenitor, A2/HEK,the cpts530/1030 mutant differs in seven amino acids (four in L, two inF, and one in N). TABLE 37 Nucleotide Sequence Differences among cp-RSV,cpts-530, cpts- 530/1009 mutant viruses Nucleotide and Amino Acid ChangeAmino A2 wt cpts- cpts- Nucleotide¹ Acid Gene (HEK-7) cp-RSV 530530/1009 1938 267 N G (val) A (ile) A (ile) A (ile) 6313 218 F A (glu) C(asp) C (asp) C (asp) 7228 523 F C (thr) T (ile) T (ile) T (ile) 9453319 L G (cys) A (tyr) A (tyr) A (tyr) 10060 521 L C (phe) C (phe) A(leu) A (leu) 12002 1169 L A (met) A (met) A (met) G (val) 13565 1690 LC (his) T (tyr) T (tyr) T (tyr)¹Positive sense

Thus, the ts and attenuation phenotypes of the cpts248 and cpts530 eachare associated with a single nucleotide change in the polymerase gene.The incremental increase in ts and attenuation phenotypes between thecpts530, and cpts530/1009 or cpts530/1030 was also each associated witha one amino change in L. In these four examples (i.e., cpts248, cpts530,cpts530/1009, and cpts530/1030) a single, but different, amino acidsubstitution in L conferred the ts and attenuation phenotypes on theprogenitor strains. These amino acid substitutions are acting incooperation with the five cpRSV mutations to enhance the stability ofthe ts phenotype following replication in animals. The incrementalincrease in attenuation and temperature sensitivity observed between thecpts248 and cpts248/404 was associated with two nucleotide changes,either or both of which could contribute to the ts and attenuationphenotypes. The four specific sites in L (i.e., those specific for thecpts530, cpts530/1009, cpts530/1030 and cpts248 viruses) that are singlyassociated with the ts and attenuation phenotypes and one or both sitesin cpts248/404 are identified by the findings summarized herein as coreregions of the RSV genome or L protein at which mutation can lead toattenuation. Although the specific mutations at the four sites in the Lprotein were specific amino acid substitutions, it is likely that otheramino acid substitutions as well as in frame insertions or deletions atthese sites and at contiguous amino acids within about five amino acidsof a specific site can also result in attenuation.

The encoded amino acid changes in L do not appear to involve the regionsof highest sequence conservation among the paramyxovirus polymeraseproteins, the proposed ATP binding site (Stec et al., Virology183:273-287 (1991)), nor the regions suggested to be homologous tomotifs of RNA-dependent RNA and DNA polymerases (Poch et al., EMBO J.8:3867-3874 (1989)). It is more likely that the effect of thesemutations is at amino acid level rather than nucleotide level, giventhat the mutation does not lie within the 3′ and 5′ genome termini northe short gene-start and gene-end sequences. These RNA regions arethought to contain all of the cis-acting RNA sequences required forefficient encapsidation, transcription, replication, and packaging intovirions (Collins et al., Proc. Natl. Acad. Sci. USA 88:9663-9667 (1991);Collins et al., Proc. Natl. Acad. Sci. USA 93:81-85 (1996), eachincorporated herein by reference).

These results provide the first full-length sequence of a tsRSV mutant.These results also indicate that pneumoviruses can be attenuated by thesubstitution of a single nucleotide that causes an amino acid change orchange in, e.g, a GS sequence. Since the cpts248 and cpts530 viruseshave a high degree of stability of the ts phenotype both in vitro and invivo, it is remarkable indeed that this phenotype was found to beassociated with a single, different amino acid change. Importantly, thecpts248/404, cpts530/1009, and cpts530/1030 contain at least threemutations that contribute to the attenuation phenotype, two ts and onenon-ts (e.g., the five cp mutations), and this is a partial non-limitingexplanation for the high level of stability of these viruses in vitroand in vivo.

Determination of the complete sequence of RSV vaccine virus strains andof their parental viruses permits analysis at the genetic level of thestability of vaccine viruses during vaccine virus production and duringshedding by volunteers in clinical trials. The determination of thegenetic basis for the attenuation and ts phenotypes of the cpts248,cpts530, cpts248/404 cpts530/1009 and cpts530/1030 viruses providesimportant new opportunities. According to the recombinant methodsdescribed hereinbelow, it is readily possible to generate novel vaccinecandidates by site-directed mutagenesis of full-length RSV cDNA fromwhich infectious viruses can be recovered. For example, it is possibleto add to a cDNA clone of, e.g., the cpts248/404 virus, one or both ofthe ts mutations at amino acid position 521 (in the cpts530 mutant) or1169 (in the cpts530/1009 mutant) or other attenuating or stabilizingmutations as desired. In this way, the level of attenuation of thecpts248/404 virus can be increased in an incremental fashion and avaccine strain that has the specific level of attenuation desired forboth safety and immunogenicity can be generated in a rational way.Similarly, the level of attenuation of the cpts530/1009 and cpts530/1030mutants can be increased by the specific introduction of one or more ofthe attenuating mutations in the cpts248/404 virus. These examples ofcombinatorial recombinant viruses, incorporating multiple attenuatingmutations from biologically derived mutant strains, overcome many of thedifficulties which attend isolation and production of geneticallystable, satisfactorily attenuated viruses using conventional approaches.Moreover, the phenotypic stability of these recombinant cptsRSV mutantscan be enhanced by introducing, where possible, two or more nucleotidesubstitutions at codons that specify specific amino acids that are knownto confer the attenuation phenotype. In this way the stability of theattenuation phenotype can be augmented by site-directed mutagenesis offull-length RSV cDNA.

EXAMPLE VII Construction of cDNA Encoding RSV Antigenome

A cDNA clone encoding the antigenome of RSV strain A2 was constructed,as illustrated in FIG. 2. The cDNA was synthesized in segments byreverse transcription (RT) and polymerase chain reaction (PCR) usingsynthetic oligonucleotides as primers and intracellular RSV mRNA orgenome RNA isolated from purified virions as template. The final cDNAwas flanked on the leader end by the promoter for T7 RNA polymerase,which included three transcribed G residues for optimal activity;transcription would result in the donation of these three nonviral G'sto the 5′ end of the antigenome. To generate a nearly-correct 3′ end,the cDNA trailer end was constructed to be adjacent to apreviously-described hammerhead ribozyme, which upon cleavage woulddonate a single 3′-phosphorylated U residue to the 3′ end of the encodedRNA (Grosfeld et al., J. Virol. 69:5677-5686 (1995), incorporated hereinby reference). The ribozyme sequence was followed by a tandem pair ofterminators of T7 RNA polymerase. (The addition of three 5′ G residuesand one 3′ U residue to a cDNA-encoded RSV minigenome containing thechloramphenicol acetyl transferase (CAT) reporter gene had no effect onthe expression of CAT when complemented by RSV.)

FIG. 2 shows the structures of the cDNA and the encoded antigenome RNA.The diagram of the antigenome (at top) includes the following features:the 5′-terminal nonviral G triplet contributed by the T7 promoter, thefour sequence markers at positions 1099 (which adds one nt to thelength), 1139, 5611, and 7559, the ribozyme and tandem T7 terminators,and the single nonviral 3′-phosphorylated U residue contributed to the 3end by ribozyme cleavage (the site of cleavage is indicated with anarrow).

Cloned cDNA segments (FIG. 2, middle) representing in aggregate thecomplete antigenome were constructed by RT-PCR of RSV mRNA or genomeRNA. The complete antigenome cDNA is called D46 or D53; the differentnames referring to different preparations of the same plasmid. cDNAscontaining the lefthand end of the antigenome, spanning from the T7promoter and leader region complement to the SH gene and called D13,were assembled in a version of pBR322 (FIG. 2, bottom) in which thenaturally-occurring BamHI site had been ablated by mutagenesis and thePstI-EcoRI fragment replaced with a synthetic polylinker containingunique restriction sites (including BstBI, BstXI, PacI, BamHI, MluI)designed to facilitate assembly. The box in FIG. 2 shows the removal ofthe BamHI site. The naturally occurring BamHI-SalI fragment (the BamHIsite is shown in the top line in positive sense, underlined) wasreplaced with a PCR-generated BglII-SalI fragment (the BglII site isshown in the bottom line, underlined; its 4-nt sticky end [italics] iscompatible with that of BamHI). This resulted in a single nt change(middle line, underlined) which was silent at the amino acid level.These modifications to the vector facilitated construction of the cDNAby rendering unique a BamHI site in the antigenome cDNA.

The G, F and M2 genes were assembled in a separate plasmid, as were theL, trailer and flanking ribozyme and tandem T7 transcriptionterminators. The G-to-M2 piece was then inserted into the PacI-BamHIwindow of the leader-to-SH plasmid. This in turn was the recipient forthe is L-trailer-ribozyme-terminator piece inserted into the BamHI toMluI window, yielding the complete antigenome.

Four restriction site markers (FIG. 3) were introduced into theantigenome cDNA during the original construction by incorporating thechanges into oligonucleotide primers used in RT-PCR. This was done tofacilitate assembly, provide a means to identify recombinant virus, andillustrate the ability to introduce changes into infectious RSV. Threesites were in intergenic regions and the fourth in a nontranslated generegion, and they involved a total of five nt substitutions and a singlent insertion. This increased the length of the encoded antigenome by onent from that of wild type to a total of 15,223 nt (SEQ ID NO:1, whichdepicts the 5′ to 3′ positive-sense sequence of D46, whereas the genomeitself is negative-sense; note that position four can be either G or C).

The sequence markers were inserted into the cDNA-encoded antigenome RNAas shown in FIG. 3. Sequences are positive sense and numbered relativeto the first nt of the leader region complement as 1; identities betweenstrains A2 and 18537 (Johnson and Collins, J. Gen Virol. 69:2901-2906(1988), incorporated herein by reference), representing subgroups A andB, respectively, are indicated with dots; sequences representingrestriction sites in the cDNA are underlined; GS and GE transcriptionsignals are boxed; the initiation codon of the N translational openreading frame at position 1141 is italicized, and the sequence markersare shown underneath each sequence. In the top sequence, a single Cresidue was inserted at position 1099 to create an AflII site in theNS2-N intergenic region, and the AG at positions 1139 and 1140immediately upstream of the N translational open reading frame werereplaced with CC to create a new NcoI site. In the middle sequence,substitution of G and U at positions 5612 and 5616, respectively,created a new StuI site in the G-F intergenic region. And, in the bottomsequence of FIG. 3, a C replacement at position 7560 created a new SphIsite in the F-M2 intergenic region.

All cDNAs were sequenced in their entirety, in most instances fromseveral independent cDNAs, prior to assembly. The plasmids encodingindividual RSV proteins are described in Grosfeld et al., J. Virol.69:5677-5686 (1995) and Collins et al., supra, (1995), each of which isincorporated herein by reference. The complete cDNA was also sequencedin its entirety following assembly

EXAMPLE VIII Transfection and Recovery of Recombinant RSV

The method of the invention for producing infectious RSV fromcDNA-expressed antigenome involves its coexpression with those RSVproteins which are sufficient to (i) produce an antigenome nucleocapsidcapable of RNA replication, and (ii) render the progeny genomenucleocapsid competent for both RNA replication and transcription.Transcription by the genome nucleocapsid provides all of the other RSVproteins and initiates a productive infection.

Plasmid-borne cDNA encoding the antigenome was transfected, togetherwith plasmids encoding proteins N, P, L and M2(ORF1), into HEp-2 cellswhich had been infected with a recently-described vaccinia virus MVAstrain recombinant which expresses the T7 RNA polymerase (Wyatt et al.,Virol. 210:202-205 (1995), incorporated herein by reference). The MVAstrain is a host range mutant which grows permissively in avian cellswhereas in mammalian cells there is a block at a late stage in virionmaturation that greatly reduces the production of infectious virus. InHEp-2 cells, the MVA recombinant was similar to the more commonly-usedWR-based recombinant (Fuerst et al., Proc. Natl. Acad. Sci. USA 83:8122-8126 (1986)) with regard to the level of expression of T7polymerase and cytopathogenicity, but the level of progeny produced wassufficiently low that supernatants could be passaged to fresh cells withminimal cytopathogenicity. This should facilitate the recovery of anyrecombinant RSV which might be produced in transfected, vacciniavirus-infected cells.

Transfection and recovery of recombinant RSV was performed as follows.Monolayer cultures of HEp-2 cells received, per single well of asix-well dish, one ml of infection-transfection medium prepared bymixing five plasmids is in a final volume of 0.1 ml opti-MEM (LifeTechnologies) medium, namely 0.4 μg each of antigenome, N and Pplasmids, and 0.1 μg each of L and M2(ORF1) plasmids. This was combinedwith 0.1 ml of Opti-MEM containing 12 μl LipofectACE (LifeTechnologies). After 15 min incubation at room temperature, this wascombined with 0.8 ml of OptiMEM containing 2% heat-inactivated fetalbovine serum and 1.5×10⁶ pfu of strain MVA vaccinia virus recombinantencoding T7 RNA polymerase (Wyatt et al., supra). This was added to thecells and replaced one day later by Opti-MEM containing 2% serum.Cultures were incubated at 32° C. and harvested on day three. Incubationat 32° C. was used because it was found that the MVA virus is slightlytemperature sensitive and is much more efficient at this lowertemperature.

Three days post-transfection clarified culture supernatants werepassaged onto fresh HEp-2 cells and overlaid with methyl cellulose (forsubsequent antibody staining) or agarose (for plaque isolation). Afterincubation for five days under methyl cellulose, the cells were fixedand stained by an indirect horseradish peroxidase method using a mixtureof three murine monoclonal antibodies to the RSV F protein followed byan anti-mouse antibody linked to horseradish peroxidase, following thegeneral procedure of Murphy et al., Vaccine 8:497-502 (1990).

Numerous RSV-like plaques were detected against a background ofcytopathogenicity that presumably was due to a low level of MVA-T7recombinant virus. The plaques contained an abundant amount of the RSV Fprotein, as indicated by brown-black coloration, and displayedcytopathic effects characteristic of RSV, notably syncytium formation.

The RSV-like plaques were picked from plates which were incubated underagarose and stained with neutral red. They were propagated and comparedto a laboratory strain of RSV strain A2 by plaque assay and antibodystaining. The plaques derived from the transfected cultures closelyresembled those of the laboratory strain. One difference was that theplaques derived from the transfected cultures appeared to be slightlysmaller than those from the laboratory strain, with centers which wereless well cleared. The recombinant virus may differ phenotypically fromthis particular wild type isolate, possibly being slightly morerestricted in cell-to-cell spread and exhibiting a reduced rate of cellkilling. With regard to the propagation of released virus, the yields ofthe recombinant versus laboratory virus in HEp-2 cells were essentiallyidentical at 32° or 37° C. In preliminary studies, the recombinant andlaboratory viruses were indistinguishable with regard to theaccumulation of intracellular RSV mRNAs and proteins.

Plaque-purified, thrice-passaged recombinant RSV was analyzed inparallel with laboratory virus by RT-PCR using three primer pairsflanking the four inserted markers. Three independent plaque-purifiedrecombinant RSV isolates were propagated in parallel with an uninfectedcontrol culture. Clarified medium supernatants were treated withpolyethylene glycol and high salt (Zoller and Smith, DNA 3:479-488(1984)) to precipitate virus and RNA was extracted from the pellets withTrizol™ (Life Technologies). These RNAs, in parallel with additionalcontrols of no added RNA or 0.1 μg of RNA from a laboratory isolate ofstrain A2, were treated with DNAse, repurified, annealed each with 50 ngof random hexamers and incubated under standard RT conditions (40 μlreactions) with or without reverse transcriptase (Connors et al., Virol.208: 478-484 (1995), incorporated herein by reference). Aliquots of eachreaction were subjected to PCR (35 cycles of 94° C. for 45 s, 37° C. for30 s, 72° C. for 1 min) using three different pairs of syntheticdeoxyoligonucleotide primers. Primer pair (A): positive-sense, positions925-942 and negative-sense, positions 1421-1440, yielding a predictedproduct of 516 bp (517 bp in the case of the recombinant viruses) thatincluded the AflII and NcoI sites inserted at, respectively, thejunction of the NS2 and N genes and in the N gene. Primer pair (B):positive-sense, positions 5412-5429 and negative-sense, 5930-5949,yielding a predicted product of 538 bp spanning the StuI site insertedat the junction between the G and F genes. Primer pair (C):positive-sense, 7280-7297 and negative-sense, 7690-7707, yielding a 428bp fragment spanning the SphI site inserted at the junction between theF and M2 genes. PCR products were analyzed by electrophoresis on neutralgels containing 1% agarose and 2% low-melting agarose in parallel withHaeIII-digested X174 DNA molecular length markers and visualized bystaining with ethidium bromide. PCR products of the expected sizes wereproduced. The production of each was dependent on the RT step,indicating that each was derived from RNA rather than contaminatingcDNA.

PCR products were analyzed by digestion with restriction enzymes.Digestion of products of primer pair A with AflII or NcoI yieldedfragments corresponding to the predicted 177 and 340 bp (AflII) or 217and 300 bp (NcoI). Digestion of products of primer pair B with StuIyielded fragments comparable to the predicted 201 and 337 bp. Digestionof products from reactions with primer pair C with SphI yielded productscorresponding to the predicted 147 and 281 bp. The digests were analyzedby gel electrophoresis as above. The presence of residual undigested PCRproduct with AflII was due to incomplete digestion, as was confirmed byredigestion. Thus, the restriction enzyme digestion showed that the PCRproducts representing recombinant virus contained the expectedrestriction site markers while those representing the laboratory straindid not. Nucleotide sequence analysis of cloned PCR product confirmedthe sequences spanning the restriction site markers.

As shown in Table 38, the efficiency of RSV production when complementedby N, P, L and M2(ORF1) was relatively high, ranging in threeexperiments from an average of 9.9 to 94.8 plaques per 0.4 μg of inputantigenome cDNA and 1.5×10⁶ cells. Since these plaques were derived fromliquid overlay, the number of infected cells present in each well of theoriginal transfection was not known. Nearly every transfected well (54of 56 in Table 38) produced virus. Since the yield of released RSV perinfected cell typically is very low (˜10 pfu) even under idealconditions, and since many wells yielded many times this amount (up to169 plaques), it is likely that several RSV producing cells were presentin many of the wells of transfected cells.

RSV was not recovered if any of the plasmids were omitted, as shown inTable 38. The requirement for M2(ORF1) also could be satisfied with thecomplete gene, M2(ORF1+2), provided the level of its input cDNA was low(0.016 μg per 1.5×10⁶ cells (Table 38). At higher levels, the productionof virus was greatly reduced, suggesting that an inhibition ofminigenome RNA synthesis associated with M2(ORF2) also operates on thecomplete genome during productive infection.

These results showed that the production of infectious RSV was highlydependent on expression of the M2(ORF1) protein in addition to N, P andL. Furthermore, it showed that the optimal method of expression ofM2(ORF1) was from an engineered cDNA in which ORF2 had been deleted,although the complete cDNA containing both ORFs also supported theproduction of RSV.

Thus, the present invention demonstrates that transcription by RSVdiffers from that of from previously-described nonsegmented negativestrand RNA viruses in requiring a fourth protein designated here asM2(ORF1), and previously called 22K or M2 (Collins et al., J. Virol.54:65-71 (1985)). The M2(ORF1) protein was found to be an RNA polymeraseelongation factor essential for processive, sequential transcription.Collins et al., Proc. Natl. Acad. Sci. USA 93:81-85 (1996). Thisrequirement provides the capability, as part of this invention, forintroducing specific, predetermined changes into infectious RSV. TABLE38 Production of infectious RSV was dependent on expression of M2 ORF 1.Complementing plasmids (μg cDNA per 1.5 × 10⁶ Production of infectiousRSV cells and antigenome # plaques × # wells* cDNA) 0.4 μg expt. 1 expt.2 expt. 3 N(0.4) P(0.4)  0 × 24  0 × 12  0 × 12 L(0.1) N(0.4)   0 ×19^(§) 0 × 4  9 × 1 P(0.4) 1 × 2 3 × 1 10 × 1 L(0.1) 2 × 2 5 × 1 14 × 2M2 [ORF1 + 2] (0.016) 3 × 1 6 × 1 22 × 1 9 × 1 28 × 1 av. 0.38 10 × 1 32 × 1 13 × 1  49 × 1 34 × 1  70 × 2 51 × 1  166 × 1  169 × 1  av. 10.9av. 48.6 N(0.4) 0 × 1 11 × 1 0 × 1 55 × 1 P(0.4) 1 × 1 12 × 1 2 × 1 59 ×1 L(0.1) 2 × 2 13 × 1 4 × 1 65 × 1 M2 [ORF1] (0.1) 3 × 2 21 × 1 5 × 1 71× 1 4 × 1 24 × 1 8 × 2 72 × 1 5 × 2 26 × 1 10 × 3  87 × 1 6 × 4 30 × 219 × 1  97 × 1 7 × 2 33 × 2 20 × 1  100 × 1  9 × 1 42 × 1 23 × 1  109 ×1  10 × 2  73 × 1 128 × 1  av. 9.9 147 × 1  av. 13.7 148 × 1  av. 94.8*Supernatants from transfected cultures (10⁶ cells per well) werepassaged onto fresh HEp-2 cells, overlaid with methyl cellulose, andstained with F-specific monoclonal antibodies.^(§)Read as follows: 19 wells had 0 plaques, 2 wells had 1 plaque each,2 wells had 2 plaques each, and 1 well had 3 plaques.

EXAMPLE IX Construction of an Infectious Recombinant RSV Modified toIncorporate Phenotype-Specific Mutations of RSV Strain cpts530

This Example illustrates the introduction of specific predeterminedmutations into infectious RSV using the recombinant methods describedherein. As noted above, the complete nucleotide sequence of cpts530 RSVwas determined and 5 mutations known to be present in the parent cpRSVwere retained in cpts530, the further attenuated derivative. Oneadditional nucleotide change was identified at nucleotide (nt) position10060, which resulted in a phenylalanine to leucine change at amino acidposition 521 in the large polymerase (L) protein (see Tables 37, 39).This single amino acid substitution was introduced alone or incombination with the cp mutations into the full-length cDNA clone ofwild-type A2 RSV. Analysis of infectious viruses recovered from mutantcDNAs indicated that this single mutation specified complete restrictionof plaque formation of recombinant cp530 in HEp-2 cell monolayercultures at 40° C., and the level of temperature sensitivity was notinfluenced by the presence of the 5 cpRSV mutations. These findingsidentify the phenylalanine to leucine change at amino acid position 521in the L protein as the mutation that specifies the ts phenotype ofcpts530. Similarly, one additional nucleotide change was identified inthe cpts530/1009 recombinant in comparison to its cpts530 parent virus(Table 37). This nucleotide substitution at position 12002 resulted inan amino acid change in L at position 1169 at which a methionine in thewild type virus was replaced by a valine in the cpts530/1009 mutant.This mutation has also been introduced into recombinant RSV, and therecovered virus was temperature sensitive. These findings identify themethionine to valine change at position 1169 as the mutation thatspecifies the greater level of temperature sensitivity of cpts530/1009over that of cpts530.

The levels of temperature sensitivity among the 530, 530/1009 and530/1030 recombinant virus have been confirmed with RSV-CAT orRSV-Luciferase minigenome (see above) monitored by enzyme assay orNorthern blot analysis. For example, at the elevated temperature of 37°C., the luciferase activity generated by selected mutants relative towild-type L protein was 18.4%, 1.5% and 0.4% for 1009, 530, and thedouble mutant pTM1-L support plasmid. These exemplary mutations alsodecreased L function at 32° C. with 70% activity for 1009, 40% activityfor 530, and 12.5% activity for 530/1009 L protein compared to wt Lprotein. The effects of these mutations on transcription and replicationcan also be determined using the minigenome system, alone or incombination with the recombinant viral methods disclosed herein. TABLE39 Mutations introduced into the RSV full-length cDNA clone. SequenceSequence of Restriction Amino Acid Mutation of wt Mutation Site Changesite (L)¹ ₉₃₉₈CTTAAGA ₉₃₉₈ C C TAAG G Bsu36I — site (L) ₁₁₈₄₆TACATA₁₁₈₄₆ TAC G TA SnaBI — site (L) ₁₃₃₃₉GTCTTAAT ₁₃₃₃₉ GT T T A AA C PmeI —site (L) ₁₄₀₈₂CGTACAG ₁₄₀₈₂ CG G AC C G RsrII — site (L) ₁₄₃₁₈ TGTAACA₁₄₃₁₈ G GTAAC C BstEI — site (L) ₁₄₄₇₅TATGTA ₁₄₄₇₅ TA C GTA SnaBI — HEK(F) ₅₈₄₈AATATCAAG A AA ₅₈₄₈ AATAT T AAG G *AA SspI ₆₆lys→glu HEK (F)₅₉₅₆AGCAC A C A A ₅₉₅₆ AG T AC T C C *A ScaI ₁₀₁gln→pro cp (F) ₁₉₃₅ATCAG TT ₁₉₃₅

ClaI ₂₆₇val→ile cp (F) ₆₃₁₁TA G AA A ₆₃₁₁

NruI ₂₁₈glu→ala cp (F) ₇₂₂₈ A CA AAT ₇₂₂₈

AseI ₅₂₃thr→ile cp (L) ₉₄₅₃

₉₄₅₃ T A * C ATAC lose AccI ₃₁₉cys→tyr cp (L) ₁₃₅₅₅TATTAACTAAA C A T₁₃₅₅₅T G TTAACTAAA T *A C HpaI ₁₆₉₀his→tyr 248 (L) ₁₀₉₈₂ TCATGCTC AA₁₀₉₈₂ G CATGC TC T * G SphI ₈₃₁gln→leu 404 (M2) ₇₆₀₆ TATGTCACGA ₇₆₀₆C*ATGTC G CGA NruI — 404 (L) ₁₂₀₄₂ TTG GA T ₁₂₀₄₂

XhoI ₁₁₈₃asp→glu 530 (L) ₁₀₀₅₉ TT C ₁₀₀₅₉ TT A * — ₅₂₁phe→leu 1009 (L)₁₁₉₉₂CCACTGAGATG A T G ₁₁₉₉₂

BsfXI ₁₁₆₉met→val 1030 (L) ₁₂₄₅₂ G T TAACA T AT ₁₂₄₅₂GCTAACA A *AT loseHpaI ₁₃₂₁tyr→asnNote:Nucleotide differences between wild-type and mutants (at thecorresponding positions of are underlined. Recognition sites ofrestriction endonucleases are in italics. Codons in which the introducednucleotide change(s) results in amino acid substitution are in bold.Asterisk identifies the single nucleotide change that was present in thebiologically-derived mutant virus. Numbering system reflects the onenucleotide insertion in the full length cDNA.¹Indicates the gene into which the mutation was introduced.

To incorporate the cpts530 and the cpts1009 specific mutations into adefined, attenuated recombinant RSV vaccine virus, the cDNA-basedrecovery system described herein was employed as follows. Thepreviously-described RSV A2 wild-type full-length cDNA clone (Collins etal., supra., (1995)) was designed in the original construction tocontain a single nucleotide insertion of C in the cDNA clone at ntposition 1099 (which creates an AflII site) and a total of 6 additionalnucleotide substitutions at 4 loci. Thus, the nucleotide numberingsystem for the naturally occurring virus and for the recombinant virusesderived from cDNA are out of register by one nt after position 1099. Thenucleotide numbering in Tables 36 and 37, above, represents thepositions for the naturally occurring viruses, while those for the cDNAclones (Table 39) and recombinant viruses derived from cDNA clones areone nucleotide more. One of the 6 nucleotide substitutions is a G to Cchange in genome sense at nt position 4 in the leader sequence. Thisnucleotide variation has been detected in non-recombinant RSV and wasnot found to have an effect on the temperature sensitivity of virusreplication in tissue culture or in mice (Firestone, et al., Virology,225:419-522 (1996), incorporated herein by reference). Table 39 liststhe various mutations which were inserted into the RSV cDNA in this andsubsequent Examples.

Intermediate clones (D50 and D39) were used to assemble the full-lengthRSV cDNA clone D53, which encodes positive-sense RSV antigenome (FIG.4). The D50 plasmid contains the RSV genome from the leader to the M2-Loverlap downstream of a T7 promoter, while the D39 plasmid encodes thefull-length L gene and the trailer followed by the hammerhead ribozymeand two T7 terminators (approximately 7 kb in length) bordered by BamHIand MluI restriction sites. The full-length RSV cDNA clone (D53) used intransfections to rescue infectious virus was assembled by inserting theBamHI-MluI fragment of the D39 plasmid into the D50 plasmid (see U.S.patent application Ser. No. 08/720,132; and published PCT ApplicationNo. PCT/US96/15524, each incorporated herein by reference). D50 wasfurther separated into several pieces each placed in a phagemid plasmidfor the purposes of facilitating mutagenesis: one piece was anXbal-EcoRI fragment containing the N gene (cDNA pUC118.D50N), and onewas a StuI-BamHI fragment containing the F and M2 genes (pUC118.F-M2).D39 was further separated into two pieces each placed in a separatephagemid plasmid: one piece (left hand half, cDNA pUC119.L1) runs fromthe BamHl site to the PmlI site at nucleotide 12255 (note that thesequence positions assigned to restriction site locations here andthroughout are intended as a descriptive guide and do not aloneprecisely define all of the nucleotides involved), and the other (righthand half, cDNA pUC119.L2) from the Pm/I site to the end of the T7terminator.

Mutations were placed into the pUC118- and pUC119-based constructsillustrated in the bottom row of FIG. 4 following standard procedures(see, e.g., Kunkel et al., Methods Enzymol, 54:367-382 (1987),incorporated herein by reference). The plasmids were propagated in a dutung strain of E. coli., in this case CJ236, and single stranded DNA wasprepared by infection with a helper phage, in this case M13KO7.Phosphorylated synthetic oligonucleotides each containing one or morenucleotide changes of interest were prepared, annealed to the singlestranded template singly or in combination, and used to direct DNAsynthesis by T4 DNA polymerase. The products were ligated andtransformed into a non-dut ung strain of E. coli, in this case DH5alphaor DH10B. Miniprep DNA of the transformant colonies was screened for thepresence of the mutation by restriction enzyme digestion or bynucleotide sequence analysis of the mutagenized region. Fragmentscontaining the appropriate mutations were transferred from the pUCconstructs back into the D50 or D39 plasmids, which in turn wereassembled into a full-length clone designated D53. Recombinant virus wasrecovered in HEp-2 cells by complementing the D53 plasmid with a mixtureof the four support plasmids encoding the N, P, L and M2 (ORF1)proteins, as described above.

Four types of mutations are involved in this and subsequent examplesdescribing specific recombinant RSV incorporating biologically derivedmutations (see Tables 36, 37, 39):

(1) The first group of mutations involves six translationally silent newrestriction site markers introduced into the L gene, collectively calledthe “sites” mutations. The six sites are Bsu36I, SnaBI, PmeI, RsrII,BstEII and a second, downstream SnaBI site, and are underlined in FIG. 4above the D53 diagram. These six changes, collectively referred to asthe “sites” mutations, were inserted for the purpose of facilitatingcDNA construction. Also, it is known that recombination can occur duringtransfection between the D53 plasmid and the support plasmids, i.e., theN, P, M2(ORF1) and L plasmids (Garcin et al., EMBO J. 14:6087-6094(1995), incorporated herein by reference. These restriction sites in Lare present in the D53 construct but not in the L support plasmid, andthus provide a marker to confirm that recombination in L did not occur.This is particularly important since many of the attenuating mutationsoccur in L.

(2) The second group of mutations involves two amino acid changes in theF gene (FIG. 4, Table 39). The cpRSV, and hence all of its derivatives,is derived from a wild type virus called HEK-7. The sequence of theoriginal D53 cDNA differs from that of HEK-7 by single nucleotidesubstitutions at seven positions. One is at nucleotide 4, which is a C(in negative sense) in the original D53 and G in the HEK virus. However,biologically-derived viruses have been shown to contain eitherassignment, and can fluctuate between the two, and so this difference isconsidered incidental and not considered further here. Four othernucleotide substitutions were silent at the amino acid level; these weretwo changes in F, at positions 6222 and 6387, one in the F-M2 intergenicregion at position 7560, and one in L at position 10515). These also arenot considered significant and are not considered further. Finally,there were two nucleotide substitutions, each in the F gene, which eachresulted in an amino acid substitution (Table 39). These two changes,collectively called the “HEK” assignments, were introduced into D53 suchthat the encoded recombinant wild type virus would be identical at theamino acid level to the HEK-7 wild type parent of thebiologically-derived cpts mutants. Note that each of these changes wasdesigned to also introduce a new restriction enzyme recognition site forthe purpose of monitoring the presence of the introduced mutation incDNA as well as in recovered virus.

(3) The third group of mutations involved the five amino acidsubstitutions found in the cpRSV virus, collectively called the “cp”mutations. These are present in all of the biologically-derived cptsviruses and contribute to the attenuation phenotype. Inbiologically-derived cpRSV, each of these amino acid changes is due to asingle nucleotide change. As shown in Table 39, when the amino acidcoding change was introduced into cDNA to make recombinant virus in fourof the five cases each coding change was made to involve two nucleotidesubstitutions, which renders the recombinant RSV highly resistant toreversion to wild type. Note that four of these changes were designed tointroduce a new restriction enzyme site, whereas the fifth was designedto ablate an existing site, thus providing a method for monitoring thepresence of the mutation by the presence or absence of the restrictionsite in cDNA or RT-PCR products generated from recombinant viruses.

(4) The fourth group of mutations involves point mutations specific toindividual, biologically derived cpts viruses (Table 39, FIG. 4), whichare named after the biological step at which they were acquired. Forexample, derivation of the cpts248/404 virus from cpRSV in the followingExample involved two steps of mutagenesis. The first yielded the cpts248virus, which sustained a single amino acid change that is thereforecalled the 248 mutation. The second mutagenesis step, applied tocpts248, yielded the cpts248/404 virus, which contains an amino acidchange in L called the 404(L) mutation and a nucleotide change in thegene start (GS) signal of M2, called 404(M2) mutation. The remainingmutations, namely 530, 1009 and 1030, each involve a single (different)amino acid change. The 404(M2) mutation is noteworthy because itinvolves the GS transcription signal (and does not involve aprotein-coding sequence) and because this mutation was shown in aminigenome system to be important for synthesis of the mRNA. Kuo et al.,J. Virol. 71:4944-4953 (1977) (incorporated herein by reference). Asoutlined in Tables 36, 37, and 39, the amino acid coding changes of the248, 404(L), and 1009 mutations were inserted into recombinant virususing two nucleotide substitutions for the purpose of improved geneticstability. Also, the 248, 404(M2), 404(L), and 1009 mutations forrecombinant virus were each designed to introduce a new restriction sitefor monitoring purposes, while the 1030 mutation was designed to ablatean existing site.

In the present Example, several pUC118- and pUC119-based constructs werederived from the D50 and D39 plasmids and desired mutations wereintroduced into these constructs (FIGS. 4, 5). Fragments containing theappropriate mutations were transferred from the pUC constructs back intothe DS0 or D39 plasmids as indicated, which in turn were assembled intoa full-length clone. In this way, six different types of D53 full-lengthderivative clones were generated (FIGS. 4, 5). In FIG. 5, the D53constructs lacked the two HEK mutations in F (see FIG. 4).

Mutagenesis was performed using the Muta-Gene® Phagemid in vitroMutagenesis kit (Bio-Rad, Hercules, Calif.) as recommended by themanufacturer. The mutagenized constructs were transformed into competentE. coli DH10B (Life Technologies). Miniprep DNA of the transformantcolonies was screened for the presence of the mutation by restrictionenzyme digestion (see below) or by nucleotide sequence analysis of themutagenized region.

The six translationally silent restriction site markers, the 530mutation (₅₂₁phe→leu), and the 5 cp mutations (Table 39) were introducedinto the pUC-based constructs and subcloned into the D50 and D39plasmids as indicated in FIGS. 4 and 5. The various full-length cDNAconstructs were assembled using D50 and D39 constructs containingdifferent combinations of the above mentioned mutations.

In the final cDNA constructs, the presence of the 530 and the cpmutations were confirmed by sequence analysis, while the presence of thesilent restriction sites were determined by restriction endonucleaseanalysis. Each D53-based construct was analyzed using variousrestriction enzymes (e.g. HpaI, AccI, HindIII, PstI), and therestriction patterns of the newly generated full-length cDNA clones werecompared with that of the previously rescued wild-type full-length cDNAclone. This restriction analysis was used to determine if an insertionor deletion of 100 nt or more had occurred during the bacterialamplification of the full-length plasmids.

Transfection was performed as described previously (Collins, et al.,Proc. Natl. Acad. Sci. USA, 92:11563-11567 (1995), incorporated hereinby reference). Briefly, monolayers of HEp-2 cells were infected at anMOI of 1 with recombinant vaccinia virus MVA strain expressing T7 RNApolymerase (MVA-T7) and were transfected using LipofectACE (LifeTechnologies) with a D53 antigenomic construct plus the N, P, L and M2(ORF1) pTM1 support plasmids. On day three, supernatants (clarifiedmedium) were passaged onto fresh HEp-2 cells for amplification ofrescued virus. Virus suspensions from this first amplification wereharvested 5 days after infection and, following inoculation at variousdilutions onto monolayers of HEp-2 cells, were overlayed withmethylcellulose for plaque enumeration or with agarose for plaqueharvest and biological cloning. Plaque enumeration was performed using amonoclonal antibody-horseradish peroxidase staining procedure aspreviously described (Murphy et al., Vaccine, 8:497-502 (1990),incorporated herein by reference). The recovered recombinant viruseswere biologically cloned by three successive plaque purifications, andthen used to generate virus suspensions following two passages on HEp-2cells. The biological cloning was important to ensure a homogeneouspopulation of the recovered viruses, as recombination may arise duringthe first step of the rescue between the plasmid representing thefull-length cDNA of RSV and the support plasmids containing RSV genes(Garcin et al., EMBO J., 14:6087-6094 (1995), incorporated herein byreference). These biologically cloned and amplified virus suspensionswere used in further molecular genetic or phenotypic characterization ofthe recombinant viruses. Two biologically cloned recombinant viruseswere generated for each of the cDNA constructs (FIG. 5) except forcp_(L)530-sites and cp530-sites, for which only one biological clone wasgenerated. In each case, when there were sister clones, they wereindistinguishable on the basis of genetic and biological analysesdescribed below. A representative example of the foregoing constructscorresponding to D53-530-sites (alternatively designated A2 ts530-scl1cp, or ts530-sites) has been deposited under the terms of theBudapest Treaty with the ATCC and granted the accession number VR-2545.

The recombinant RSVs generated as described above were geneticallycharacterized to determine if they indeed contained each of theintroduced mutations. Monolayers of HEp-2 cells were infected withbiologically cloned recombinant virus and total RNA was harvested 4 to 5days post infection as described above. RT was performed using randomhexamer primers and the generated cDNA was used as template in PCR usingthe Advantage™ cDNA PCR Kit (Clontech Lab. Inc., Palo Alto, Calif.) togenerate three fragments representing almost the full-length of therecombinant RSV genomes. The PCR fragments corresponded to the RSVgenome between nt positions 1-5131, 5949-10751 and 8501-15179. Also, a544 bp fragment representing a portion of the L gene in the region ofthe 530 mutation between nt positions 9665 and 10209 was generated. Thisshort PCR fragment was used in cycle sequencing (using 71001 delta TAQ™*Cycle Sequencing Kit, USB, Cleveland, Ohio) to confirm the presence orthe absence of the 530 mutation in the recovered recombinant virus,whereas the large PCR products were used in restriction enzyme digestionto confirm the presence of the silent restriction site markers and thecp mutations which were marked with specific restriction sites.

To verify that the recombinant RSVs produced according to the abovemethods incorporated the desired phenotype, i.e., the phenotypespecified by the incorporated sequence change(s), the efficiency ofplaque formation (EOP) of the recombinant RSVs and the nonrecombinantcontrol viruses was determined. Specifically, plaque titration at 32,37, 38, 39 and 40° C. using HEp-2 monolayer cultures in temperaturecontrolled water baths was conducted, as described previously (Crowe etal., Vaccine, 11:1395-1404 (1993); Firestone, et al., Virology225:419-522 (1996), each incorporated herein by reference). Plaqueidentification and enumeration was performed using antibody staining asindicated above.

The level of temperature-sensitivity of the recombinant viruses and thewild-type and biologically derived mutant cpts530 viruses is presentedin Table 40. These data show that the introduction of the silentrestriction sites or the cp mutations does not confer a ts phenotype.This latter observation is consistent with our previous finding that thecpRSV is a ts⁺ virus (Crowe, et al., Vaccine, 12:691-699 (1994)). TABLE40 Comparison of the Efficiency of Plaque Formation^(a) of Recombinantand Biologically Derived Viruses in HEp-2 Cells at Various Temperatures.Reduction in Virus titer (log₁₀) at RSV Titer (log₁₀pfu/ml indicatedtemperature at indicated temp.)^(b) compared to that at 32° C. Virus 3238 39 40 39 40 Wild-type^(c) 5.7 5.5 5.4 5.3 0.3 0.4 r-sites 5.4 5.1 5.25.2 0.2 0.2 rcp-sites 6.1 5.7 5.7 5.7 0.4 0.4 r530 6.3 6.0 4.4 <0.71.9 >5.6 r530-sites 6.4 6.1 4.4 <0.7 2.0 >5.7 rcp_(L)530-sites 5.8 5.93.8 <0.7 2.0 >5.1 rcp530-sites 6.3 5.0 4.1 <0.7 2.2 >5.6 cpts530^(c) 6.65.8 4.4 <0.7 2.2 >5.9^(a)Efficiency of plaque formation of the various RSV strains wasdetermined by plaque titration on monolayers of HEp-2 cells undersemisolid overlay for five days at the indicated temperatures (° C.).^(b)Virus titers are the average of two tests, except for r-sites and rcp-sites where data were derived from a single test.^(c)Biologically derived control viruses.

The above findings confirm that ts phenotype of the biologically derivedcpts530 virus is specified by the single mutation identified above asbeing unique to this attenuated RSV strain. Genetic analysis of thecpts530 strain was confirmed in this context by the introduction of the530 mutation into a full-length cDNA clone of the A2 wild-type ts⁺parent virus, followed by the recovery of a ts recombinant virus bearingthe 530 mutation. Analysis of the level of temperature sensitivity ofthis and additional recombinant viruses containing the 530 and cpmutations revealed that the level of temperature sensitivity specifiedby the 530 mutation was not influenced by the five cp mutations. Thus,the methods and compositions of the invention identified the 530mutation as the ts phenotype-specific mutation which is attributed withfurther attenuation of the cpts530 virus in model hosts over that of itscpRSV parent (Crowe et al., Vaccine, 12:783-90 (1994), Crowe et al.,Vaccine, 13:847-855 (1995), each incorporated herein by reference).

In addition to the above findings, introduction of the 1009 mutation or1030 mutation into recombinant RSV, in combination with the cpts530mutation, generated recombinants whose levels of temperature sensitivitywere the same as those of the respective, biologically derivedcpts530/1009 and cpts530/1030 RSV mutants.

The above findings illustrate several important advantages of therecombinant methods and RSV clones of the invention for developing liveattenuated RSV vaccines. The insertion of a selected mutation intorecombinant RSV, as well as the recovery of mutations from the RSV A2cDNA clone were relatively efficient. The antigenome cDNA clone used inthis example had been modified in the original construction to containchanges at five different loci, involving 6 nucleotide substitutions andone nucleotide insertion. Mutagenized virus are also describedcontaining mutations at an additional twelve loci involving 24additional nucleotide substitutions. The fact that only the 530 mutationimparted a phenotype detectable in tissue culture indicates the relativeease of manipulation of this large RNA genome. Although recombinationbetween the support plasmids and the full-length clone that is mediatedby the vaccinia virus enzymes can occur (Garcin et al., EMBO J.,14:6087-6094 (1995)), its frequency is sufficiently low that each of the10 viruses analyzed here possessed the mutations present in the cDNAclone from which it was derived. Thus, it is readily feasible tointroduce further attenuating mutations in a sequential manner into RSV,to achieve a desired level of attenuation.

The demonstrated use of the present RSV recovery system for directidentification of attenuating mutations and the established success formanipulating recombinant RSV allow for identification and incorporationof other desired mutations into live infectious RSV clones. Previousfindings from clinical studies and sequence analysis of the cpRSV virussuggest that the set of five non-ts mutations present in cpRSV areattenuating mutations for seropositive humans (Connors et al., Virology,208:478-84 (1995), Firestone, et al., Virology, 225:419-522 (1996),Friedewald et al., JAMA, 203:690-694 (1968), Kim et al., Pediatrics,48:745-755 (1971), each incorporated herein by reference). These andother mutations are selected for their confirmed specificity forattenuated and/or ts phenotypes using the methods described here, andcan then be assembled into a menu of attenuating mutations. These andother attenuating mutations, both ts and non-ts, can then be introducedinto the RSV A2 wild-type virus to produce a live attenuated virusselected for a proper balance between attenuation and immunogenicity. Inthis regard, it is advantageous that the 530 and other identified tsmutations are not in the G and F glycoproteins which induce theprotective immune responses to RSV in humans. This permits thedevelopment of an attenuated RSV cDNA backbone with mutations outside ofF and G that can serve as a cDNA substrate into which the F and Gglycoproteins of RSV subgroup B or those of an epidemiologicallydivergent subgroup A strain can be substituted for the A2 F and Gglycoproteins. In this way, a live attenuated RSV vaccine can be rapidlyupdated to accommodate antigenic drift within subgroup A strains, and asubgroup B vaccine component can also be rapidly produced.

EXAMPLE X Construction of an Infectious Recombinant RSV Modified toIncorporate Phenotype-Specific Mutations of RSV Strain cpts248/404

This Example illustrates additional designs for introducingpredetermined attenuating mutations into infectious RSV employing therecombinant procedures and materials described herein.

Previous sequence analysis of the RSV A2 cpts248 mutant also identifieda single mutation in the L gene, a glutamine to leucine substitution atamino acid position 831 (Table 39; Crowe et al., Virus Genes, 13:269-273(1996), incorporated herein by reference). This mutation was confirmedby the methods herein to be attenuating and ts. Sequence analysis of thefurther attenuated RSV mutant cpts248/404 revealed two additionalmutations, a nt change in the M2 gene start sequence and an amino acidsubstitution, aspartic acid to glutamic acid at amino acid position 1183in the L protein (Table 39; Firestone, et al., Virology, 225:419-522(1996), incorporated herein by reference).

The biologically-derived attenuated RSV strain cpts248/404 wasreconstructed as a recombinant virus (rA2cp/248/404) according to theabove described methods. cDNA D53 encoding rA2cp/248/404 virus wasconstructed by insertion of the sites, HEK, cp, 248 and 404 changes(Table 39). Recombinant virus (rA2cp/248/404) was recovered, plaquepurified and amplified. The presence of the mutations in recombinantvirus was analyzed by RT-PCR of viral RNA followed by restriction enzymedigestion or nucleotide sequencing or both. The rA2cp/248/404recombinant was recovered using either the pTMLwt or pTML248/404 supportplasmid, the latter of which contains all of the mutations in L presentin the biologically derived cpts248/404 mutant (Table 39, not includingthe mutations specific to the 530, 1009, or 1030 viruses). Using thepTML248/404 as a support plasmid precludes loss of 248/404 mutationspresent in a full length clone by homologous recombination with thesupport plasmid.

Recombinant viruses were recovered from D53 DNA in which only the sitesand HEK mutations were present (rA2 in Table 41), to demonstrate thatthese changes were indeed phenotypically silent as expected. Recombinantvirus containing the sites, HEK and cp mutations was recovered (calledrA2cp in Table 41), to evaluate the phenotypes specified by the cpmutations. Also, as shown in Table 41, separate viruses were constructedcontaining the sites, HEK and cp background together with (i) the 248mutation (rA2cp/248), (ii) the two 404 mutations (rA2cp/404); and the404(M2) mutation (rA2cp/404(M2), and the 404(L) mutation (rA2cp/404(L)).

These viruses were evaluated in parallel for the ability to form plaquesin HEp-2 cells at 32° C., 36° C., 37° C., 38° C. and 39° C. Thiscomparison showed that all viruses formed plaques at 32° C., and showedthat the titers of the various virus preparations were withinapproximately three log₁₀ units of each other, which is within the rangeof experimental variation typically seen among independent preparationsof RSV. The introduction of the added “sites” and HEK mutations intowild type recombinant virus (to yield virus rA2) did not alter the viruswith regard to its ability to grow at the elevated temperatures, ascompared with biologically-derived wild type virus (virus A2 wt). Theadditional introduction of the cp mutations (to yield virus rA2cp) alsodid not alter its ability to grow at elevated temperatures. This is theexpected result, because the biologically derived cpRSV from which themutations were derived does not have the ts phenotype; its mutations areof the host range variety. The 404 mutation in L does not appear to be ats mutation since rA2cp/404(L) was not ts. However, this mutation mayotherwise prove to be attenuating, as will be determined by furtheranalysis in accordance with the methods herein. Thus, of the twospecific mutations in cpts 248/404 virus only the 404M2 mutation is ts.The further addition of the 248 and 404 mutations (to yieldrA2cp/248/404 virus) resulted in a ts phenotype that was essentiallyequivalent to its biologically-derived equivalent virus as evidenced bybeing greatly impaired in the ability to form plaques at 36° C.-37° C.,with pinpoint plaques being formed at the former temperature. Theserecombinant viruses incorporated a variety of mutations predicted tohave no effect on growth in tissue culture, and additional mutationsexpected to confer a ts phenotype. Each type of mutation yielded resultsconsistent with these expectations, demonstrating that the RSV genomecan be manipulated in a reasonably predictable way. TABLE 41 Efficiencyof Plaque Formation of Selected RSV Mutants Virus titer (log₁₀pfu/ml)off at indicated temperature (° C.) Shut- Virus¹ Transfectant 32  36 3738 39 temp. A2 wt² 5.0 4.8 5.1 4.6 4.7 >39 rA2 14-1B-1A2 4.9 4.2 4.4 4.53.9 >39 rA2cp 12-2B-1B1 5.6 5.1 4.8 3.8 3.7 >39 rA2cp/248 12-6A-1A1 5.84.8* 4.0* <0.7 <0.7 38 rA2cp/404-L 16-1A-1A1 5.4 5.2 5.1 4.9 5.0 >39rA2cp/404-M2 15-1A-1A1 4.9 <0.7 <0.7 <0.7 <0.7 36 rA2cp/404 16-4B-1B13.3 1.2* <0.7 <0.7 <0.7 36 rA2cp/248/404 32-6A-2 6.0 <0.7 <0.7 <0.7 <0.736 rA2cp/248/ 13-4B-3 5.7 4.7* 2.5* nd <0.7 37 404/530 cpts248/ 5.1 <0.7<0.7 <0.7 <0.7 36 404(WLVP)²¹Recombinant (r) viruses have been plaque purified and amplified inHEp-2 cells. Note that the stocks of recombinant andbiologically-derived viruses had not been adjusted to contain equivalentamounts of pfu/ml: the level of variation seen here is typical for RSVisolates at an# early stage of amplification from plaques. Each recombinant viruscontains the L-gene sites and the F-gene HEK mutations. The code in the“Transfectant” column refers to the transfection and plaquing history.²Biologically derived viruses: A2 wt and cpts248/404 (WLVP L16210B-150).*Pin-point plaque size

Table 41 shows further characterization of mutations specific to the 248and 404 mutagenesis steps. Specifically, viruses were constructed usingthe sites, HEK and cp background with the addition of: (i) the 248mutation (to yield virus rA2cp/248), (ii) the two 404 mutations (virusrA2cp/404); or the 404(M2) mutation (virus rA2cp/404(M2)) (Table 39).Each of these three viruses exhibited the ts phenotype, providing directidentification of these mutations as being ts. The 248 mutation provideda lower level of temperature sensitivity, whereas the 404(M2) mutationwas highly ts and was not augmented by the addition of the 404(L)mutation. It was remarkable that the 404(M2) mutation is ts, since it isa point mutation in a transcription initiation, or gene start (GS),signal, and this type of mutation has never been shown to be ts.

EXAMPLE XI Construction of Recombinant RSV Combining PredeterminedAttenuating Mutations from Multiple Attenuated Parent Viruses

The present Example illustrates a combinatorial design for producing amultiply attenuated recombinant RSV (rA2cp/248/404/530) whichincorporates three attenuating mutations from one biologically derivedRSV strain (specifically cpts248/404) and an additional attenuatingmutation from another RSV strain (cpts530). This recombinant RSVexemplifies the methods of the invention for engineering stepwiseattenuating mutations to fine tune the level of attenuation in RSVvaccines, wherein multiple mutations contribute to a further attenuatedphenotype of the vaccine strain and provide for enhanced geneticstability.

The cDNAs and methods of Examples IX and X above were used to constructa D53 cDNA containing the sites, HEK, cp, 248, 404 and 530 changes(Table 39). This involved a combination of attenuating mutations fromfour separate biologically-derived viruses, namely cpRSV, cpts248,cpts248/404 and cpts530. Recombinant virus (rA2cp/248/404/530) wasrecovered, plaque purified and amplified. The presence of the mutationswas analyzed by RT-PCR of viral RNA followed by restriction enzymedigestion or nucleotide sequencing or both. rA2cp/248/404/530 lacked the248 mutation in L, but possessed the 530 mutation and the other 248/404mutation.

The rA2cp/248/404 virus was evaluated for its ability to form plaques inHEp-2 cells at 32° C., 36° C., 37° C., 38° C. and 39° C. as describedabove (Table 41). All of viruses formed plaques at 32° C. and weresimilar to wild type in the efficiency of growth at this temperature.The rA2cp/248/404/530 virus was essentially equivalent to thebiologically-derived cpts248/404 virus with regard to the ts phenotype,evidenced by being greatly impaired in the ability to form plaques at37° C. Thus, the addition of the 530 mutation to the rA2cp/248/404background did not increase its ts phenotype, however, the recombinantlacked the 248 mutation in L.

Based on this and the foregoing Examples, the invention allows recoveryof a wide variety of recombinant viruses containing two or more tsmutations from the set of mutations which have been identified andconfirmed from biologically derived RSV mutants, for example the 248,404, 530, 1009, or 1030 biological mutants. Examples of such recombinantviruses include RSV having a combination of 248/404/530 mutations,248/404/1009 mutations, 248/404/1030 mutations, 248/404/530/1009mutations, 248/404/530/1030 mutations, 248/404/530/1030 mutations,248/404/1009/1030 mutations, or other combinations of attenuatingmutations disclosed herein. In addition, recombinant RSV incorporatingone or more ts mutations identified from biologically derived RSVmutants can be combined with other mutations disclosed herein, such asattenuating gene deletions or mutations that modulate RSV geneexpression. One such example is to combine 248/404 mutations with an SHgene deletion in a recombinant clone, which yields a viable vaccinecandidate. A host of other combinatorial mutants are provided as well,which can incorporate any one or more ts mutations from biologicallyderived RSV and any one or more other mutations disclosed herein, suchas deletions, substitutions, additions and/or rearrangements of genes orgene segments. The cp mutations, which are attenuating host range ratherthan attenuating ts mutations, also can be included along with one ormore other mutations within the invention. This provides a panel ofviruses representing a broad spectrum in terms of individual andcombined levels of attenuation, immunogenicity and genetic stability.These attenuating mutations from biologically derived RSV mutants can befurther combined with the various other structural modificationsidentified herein, which also specify desired phenotypic changes inrecombinant RSV, to yield yet additional RSV having superior vaccinecharacteristics.

EXAMPLE XII Recovery of Infectious Respiratory Syncytial VirusExpressing an Additional, Foreign Gene

The methods described above were used to construct recombinant RSVcontaining an additional gene, encoding chloramphenicol acetyltransferase (CAT). The CAT coding sequence was flanked by RSV-specificgene-start and gene-end motifs, the transcription signals for the viralRNA-dependent RNA polymerase. Kuo et al., J. Virol. 70:6892-6901 (1996)(incorporated herein by reference). The RSV/CAT chimeric transcriptioncassette was inserted into the intergenic region between the G and Fgenes of the complete cDNA-encoded positive-sense RSV antigenome, andinfectious CAT-expressing recombinant RSV was recovered. The CAT mRNAwas efficiently expressed and the levels of the G and F mRNAs werecomparable to those expressed by wild type recombinant RSV. TheCAT-containing and wild type viruses were similar with regard to thelevels of synthesis of the major viral proteins.

Plasmid D46 was used for construction of cDNA encoding RSV antigenomicRNA containing the CAT gene. (Plasmids D46 and D53, the latter beingdescribed above, are different preparations of the same antigenomecDNA.) D46 which encodes the complete, 15,223-nucleotide RSV antigenome(one nucleotide longer than that of wild type RSV) and was used toproduce recombinant infectious RSV, as described above. During itsconstruction, the antigenome cDNA had been modified to contain four newrestriction sites as markers. One of these, a StuI site placed in theintergenic region between the G and F genes (positions 5611-5616 in the3′-5′ sequence of the wild type genome), was chosen as an insertion sitefor the foreign CAT gene. A copy of the CAT ORF flanked on the upstreamend by the RSV GS signal and on the downstream end by the RS GE signalwas derived from a previously-described RSV-CAT minigenome (Collins etal., Proc. Natl. Acad. Sci. USA 88:9663-9667 (1991) and Kuo et al., J.Virol. 70: 6892-6901 (1996), incorporated by reference herein). Theinsertion of this RSV/CAT transcription cassette into the StuI siteyielded the D46/1024CAT cDNA (deposited under the terms of the BudapestTreaty with the ATCC and granted the accession number VR-2544), whichincreased the length of the encoded antigenome to a total of 15,984nucleotides. And, whereas wild type RSV encodes ten major subgenomicmRNAs, the recombinant virus predicted from the D46/1024CAT antigenomewould encode the CAT gene as an eleventh mRNA. The strategy ofconstruction is shown in FIG. 6.

Producing infectious RSV from cDNA-encoded antigenomic RNA, as describedabove, involved coexpression in HEp-2 cells of five cDNAs separatelyencoding the antigenomic RNA or the N, P, L or M2(ORF1) protein, whichare necessary and sufficient for viral RNA replication andtranscription. cDNA expression was driven by T7 RNA polymerase suppliedby a vaccinia-T7 recombinant virus based on the MVA strain. The MVA-T7recombinant virus produced infectious progeny sufficient to causeextensive cytopathogenicity upon passage, and therefore, cytosinearabinoside, an inhibitor of vaccinia virus replication, was added 24 hfollowing the transfection and maintained during the first six passages.The use of cytosine arabinoside was not required, however, and was notused in later examples herein.

Two antigenome cDNAs were tested for the recovery of RSV: the D46 cDNA,and the D46/1024CAT cDNA. Each one yielded infectious recombinant RSV.Cells infected with the D46/1024CAT recombinant virus expressed abundantlevels of CAT enzyme. For each virus, transfection supernatants werepassaged to fresh cells, and a total of eight serial passages wereperformed at intervals of five to six days and a multiplicity ofinfection of less than 0.1 PFU per cell.

The CAT sequence in the D46/1024CAT genome was flanked by RSV GS and GEsignals, and thus should be expressed as an additional, separate,polyadenylated mRNA. The presence of this predicted mRNA was tested byNorthern blot hybridization of RNA from cells infected with D46/1024CATvirus or D46 virus at the eighth passage. Hybridization with anegative-sense CAT-specific riboprobe detected a major band which was ofthe appropriate size to be the predicted CAT mRNA, which would contain735 nucleotides not including poly(A). This species was efficientlyretained by oligo(dT) latex particles, showing that it waspolyadenylated. In some cases, a minor larger CAT-specific species wasdetected which was of the appropriate size to be a G-CAT readthroughmRNA. The D46/1024CAT virus had been subjected to eight passages at lowmultiplicity of infection prior to the infection used for preparing theintracellular RNA. There was no evidence of shorter forms of the CATmRNA, as might have arisen if the CAT gene was subject to deletion.

Replicate blots were hybridized with negative-sense riboprobe specificto the CAT, SH, G or F gene, the latter two genes flanking the insertedCAT gene. The blots showed that the expression of the subgenomic SH, Gand F mRNAs was similar for the two viruses. Phosphoimagery was used tocompare the amount of hybridized radioactivity in each of the three RSVmRNA bands for D46/1024CAT and D46. The ratio of radioactivity betweenD46/1024CAT and D46 was determined for each mRNA: SH, 0.77; G, 0.87; andF, 0.78. The deviation from unity probably indicates that slightly lessRNA was loaded for D46/1024CAT versus D46, although it is also possiblethat the overall level of mRNA accumulation was slightly less forD46/1024CAT RSV. The demonstration that the three ratios were similarconfirms that the level of expression of each of these mRNAs wasapproximately the same for D46/1024CAT versus D46. Thus, the insertionof the CAT gene between the G and F genes did not drastically affect thelevel of transcription of either gene.

To characterize viral protein synthesis, infected HEp-2 cells werelabeled with [³⁵S]methionine, and cell lysates were analyzed by PAGEeither directly or following immunoprecipitation under conditions whererecovery of labeled antigen was essentially complete. Precipitation witha rabbit antiserum raised against purified RSV showed that theD46/1024CAT and D46 viruses both expressed similar amounts of the majorviral proteins F₁, N, P, M, and M2. That a similar level of M2 proteinwas recovered for each virus was noteworthy because its gene isdownstream of the inserted CAT gene. Accumulation of the F protein,which is encoded by the gene located immediately downstream of theinsertion, also was examined by immunoprecipitation with a mixture ofthree anti-F monoclonal antibodies. A similar level of the F₁ subunitwas recovered for each virus. Phosphorimagery analysis of the majorviral proteins mentioned above was performed for several independentexperiments and showed some sample-to-sample variability, but overallthe two viruses could not be distinguished on the basis of the level ofrecovered proteins. Precipitation with anti-CAT antibodies recovered asingle species for the D46/1024CAT but not for the D46 virus. Analysisof the total labeled protein showed that the N, P and M proteins couldbe detected without immunoprecipitation (although detection of thelatter was complicated by its comigration with a cellular species) andconfirmed that the two viruses yielded similar patterns. The positioncorresponding to that of the CAT protein contained more radioactivity inthe D46/1024CAT pattern compared to that of D46, as was confirmed byphosphorimagery of independent experiments. This suggested that the CATprotein could be detected among the total labeled proteins withoutprecipitation, although this demonstration was complicated by thepresence of a comigrating background band in the uninfected andD46-infected patterns.

RT-PCR was used to confirm the presence of the CAT gene in the predictedlocation of the genome of recombinant RSV. Total intracellular RNA wasisolated from the cell pellet of passage eight of both D46/1024CAT andD46 RSV. Two primers were chosen that flank the site of insertion, theStuI restriction endonuclease site at RSV positions 5611-5616: theupstream positive-sense primer corresponded to positions 5412-5429, andthe downstream negative-sense one to positions 5730-5711. Thepositive-sense primer was used for the RT step, and both primers wereincluded in the PCR.

RT-PCR of the D46 virus yielded a single product that corresponded tothe predicted fragment of 318 nucleotides, representing the G/F genejunction without additional foreign sequence. Analysis of D46/1024CATviral RNA yielded a single product whose electrophoretic mobilitycorresponded well with the predicted 1079 nucleotide fragment,representing the G/F gene junction containing the inserted CATtranscription cassette. The latter PCR yielded a single major band; theabsence of detectable smaller products indicated that the population ofrecombinant genomes did not contain a large number of molecules with adeletion in this region. When PCR analysis was performed on D46/1024CATvirus RNA without the RT step, no band was seen, confirming that theanalysis was specific to RNA. Thus, the RT-PCR analysis confirmed thepresence of an insert of the predicted length in the predicted locationin the genomic RNA of the D46/1024CAT recombinant virus.

Enzyme expression was used to measure the stability of the CAT gene.Cell pellets from all of the passages beginning with the third weretested for CAT expression. For the virus D46/1024CAT, all these assaysdisplayed conversion of [¹⁴C] labeled chloramphenicol into acetylatedforms. To investigate stability of expression, virus from 20 or 25individual plaques from passage three or eight, respectively, wasanalyzed for CAT expression. All samples were positive, and the level ofexpression of CAT was similar for each of the 25 isolates from passageeight, as judged by assay of equivalent aliquots of cell lysate. Thisdemonstrated that the activity of the CAT protein encoded by eachisolate remained unimpaired by mutation.

To determine plaque morphology and size, beginning with the secondpassage, one-eighth of the medium supernatant (i.e., 0.5 ml) harvestedfrom each passage stage was used to infect fresh HEp-2 cells in six-wellplates that were incubated under methylcellulose overlay for five toseven days. The cells were then fixed and stained by incubation withmonoclonal antibodies against RSV F protein followed by a secondantibody linked to horseradish peroxidase. Earlier, it had been observedthat recombinant RSV produced from cDNA D46 was indistinguishable from anaturally-occurring wild type RSV isolate with regard to efficiency ofplaque formation over a range of temperatures in vitro, and the abilityto replicate and cause disease when inoculated into the respiratorytract of previously uninfected chimpanzees. Thus, the D46 recombinantRSV was considered to be a virulent wild type strain. The plaquesproduced by the D46 and D46/1024CAT recombinant viruses were compared byantibody staining. Plaque morphology was very similar for the twoviruses, although the average diameter of the CAT-containing recombinantplaques was 90 percent of that of the D46 virus, based on measurement ofthirty randomly-selected plaques for each virus.

The efficiency of replication in tissue culture of the D46 andD46/1024CAT viruses was compared in a single step growth cycle.Triplicate monolayers of cells were infected with either virus, andsamples were taken at 12 h intervals and quantitated by plaque assay.The results showed that the production of D46/1024CAT virus relative toD46 was delayed and achieved a maximum titer which was 20-fold lower.

These results show that it is possible to construct recombinant,helper-independent RSV expressing a foreign gene, in this instance theCAT gene. The recombinant RSV directed expression of the predictedpolyadenylated subgenomic mRNA that encoded CAT protein, the proteinbeing detected both by enzyme assay and by radioimmunoprecipitation.Other examples have produced RSV recombinants with the luciferase geneinserted at the same CAT site, or with the CAT or luciferase genesinserted between the SH and G genes. These viruses also exhibit reducedgrowth, whereas the numerous wild type recombinant viruses recoveredexhibit undiminished growth. This indicates that the reduced growthindeed is associated with the inserted gene rather than being due tochance mutation elsewhere in the genome. The level of attenuationappears to increase with increasing length of the inserted gene. Thefinding that insertion of a foreign gene into recombinant RSV reducedits level of replication and was stable during passage in vitro suggeststhat this provides yet another means for effecting attenuation forvaccine use. Also, the insertion into recombinant RSV of a geneexpressing a protein having antiviral activity, such as gamma interferonand IL-2, among others, will yield attenuation of the virus due toactivity of the expressed antiviral protein.

In addition to demonstrating recovery of RSV having modified growthcharacteristics, the examples herein illustrate other important methodsand advantages for gene expression of recombinant RSV and othernonsegmented, negative strand viruses. For example, the data providedherein show that foreign coding sequences can be introduced as aseparate transcription cassette which is expressed as a separate mRNA.These results also show that RSV is tolerant of substantial increases ingenome length, e.g., of 762 nucleotides in the case of the CAT gene to atotal of 15,984 nucleotides (1.05 times that of wild type RSV). Theluciferase gene that was successfully recovered is almost three timeslonger.

The viral RNA-dependent RNA polymerases are known to have an error-pronenature due to the absence of proofreading and repair mechanisms. In RNAvirus genomes, the frequency of mutation is estimated to be as high as10⁻⁴-10⁻⁵ per site on average (Holland et al., Curr. Top. Microbiol.Immunol. 176:1-20 (1992) and references therein). In the case of therecombinant D46/1024CAT RSV produced here, correct expression of theforeign gene would be irrelevant for virus replication and would be freeto accumulate mutations. The passages described here involved amultiplicity of infection less than 0.1 PFU per cell, and the durationof each passage level indicated that multiple rounds of infection wereinvolved. While yields of infectious virus from RSV-infected tissueculture cells typically are low, intracellular macromolecular synthesisis robust, and the poor yields of infectious virus seem to represent aninefficient step in packaging rather than low levels of RNA replication.Thus, the maintenance of CAT through eight serial passages involved manyrounds of RNA replication. It was surprising that the nonessential CATgene remained intact and capable of encoding fully functional protein ineach of the 25 isolates tested at the eighth passage. Also, RT-PCRanalysis of RNA isolated from passage eight did not detect deletionswithin the CAT gene.

A second infectious RSV-CAT recombinant was constructed in which the CATtranscription cassette was inserted into an XmaI site which had beenengineered into the SH-G intergenic region (which is one position closerto the promoter than the F-G intergenic used above). The growthcharacteristics of this recombinant were similar to those of theD46/1024 CAT recombinant, whereas the level of CAT expression wasapproximately two to three-fold higher, consistent with its morepromoter-proximal location. This illustrates that a foreign gene can beinserted at a second, different site within the genome and its level ofexpression altered accordingly. In principle, any portion of the genomeshould be able to accept the insertion of a transcription unit encodinga foreign protein as long as the insertion does not disrupt amRNA-encoding unit and does not interfere with the cis-acting sequenceelements found at both ends of the genome.

An infectious recombinant RSV was also recovered in which the CAT genewas replaced by that of the luciferase (LUC) marker enzyme. The LUCcoding sequence is approximately 1,750 bp (three times the size of CAT),and is larger than any of the RSV genes except for F and L. It wasinserted at either the SH-G or G-F intergenic regions and infectiousrecombinant virus was recovered. The LUC viruses were further attenuatedrelative to the CAT viruses with regard to growth in tissue culture,suggesting that increases in the size of the genome lead to decreases ingrowth efficiency. Characterization of these CAT and LUC viruses willdetermine the effect of foreign gene size on virus gene expression andgrowth, such as how much is due to the introduction of the additionalset of transcription signals, and how much is due to increased genomelength.

In the minireplicon system, an RSV-CAT minigenome was modified such thatthe transcriptional unit is in the “sense” orientation in thepositive-sense antigenome rather than the minigenome. Thus, subgenomicmRNA can be made only if the polymerase is capable of transcription ofthe antigenome replicative intermediate. Interestingly, whencomplemented by plasmid-expressed N, P, L and M2 ORF1 protein, thisinverse RSV-CAT minigenome was capable of synthesizing subgenomic,polyadenylated, translatable mRNA. Efficient mRNA synthesis wasdependent on the M2 ORF1 protein, as is the case for minigenometranscription. The level of mRNA relative to its antigenome template wasapproximately the same as the ratio of mRNA to minigenome template madeby a standard minireplicon. This indicates that the antigenome, as wellas the genome, can be used to accept a foreign transcriptional unit.Thus, expression of a foreign gene can be achieved without placing itinto the genome transcriptional order. This had the advantage that theforeign gene is not be part of the transcriptional program of the genomeand thus will not perturb the relative levels of expression of thesegenes.

Because most of the antigenic difference between the two RSV antigenicsubgroups resides in the G glycoprotein, recombinant RSV can beconstructed to express the G protein of the heterologous subgroup as anadditional gene to yield a divalent vaccine. Envelope protein genes ofsome other respiratory viruses, such as human parainfluenza 3 virus,also can be inserted for expression by recombinant RSV. Other usesinclude coexpression of immune modulators such as interleukin 6 toenhance the immunogenicity of infectious RSV. Other uses, such asemploying modified RSV as described herein as a vector for gene therapy,are also provided.

EXAMPLE XIII Recombinant RSV Having a Deletion of the SH Gene

This example describes production of a recombinant RSV in whichexpression of the SH gene has been ablated by removal of apolynucleotide sequence encoding the SH mRNA and protein. The SH proteinis a small (64 amino acids in the case of strain A2) protein whichcontains a putative transmembrane domain at amino acid positions 14-41.It is oriented in the membrane with the C-terminus exposed and there isa potential glycosylation site in both the C-terminal and N-terminaldomains (Collins et al., J. Gen. Virol. 71:3015-3020 (1990),incorporated herein by reference). In infected cells, the SH protein ofstrain A2 accumulates in four major forms; (i) SH0 (Mr 7500), thefull-length, unglycosylated form that is the most abundant (Olmsted etal., J. Virol. 63:2019-2029 (1989), incorporated herein by reference);(ii) SHg (Mr 13,000-15,000), which is the full length form containing asingle N-linked carbohydrate chain; (iii) SHp (Mr 21,000-40,000), whichis a modified version of SHg in which the single N-linked carbohydratechain is modified by the addition of polylactosaminoglycan (Anderson etal., Virology 191:417-430 (1992), incorporated herein by reference);(iv) SHt (Mr 4800), a truncated unglycosylated form which is initiatedfrom the second methionyl codon (position 23) and which alone among thedifferent forms does not appear to be transported to the cell surface.The SH0 and SHp forms have been detected in purified virions, suggestingthat there is a selectivity at the level of virion morphogenesis(Collins et al., supra, (1993)).

Among the paramyxoviruses, ostensibly similar SH proteins have beenfound in simian virus 5 (Hiebert et al., J. Virol. 55:744-751 (1985),incorporated herein by reference), bovine RSV (Samal et al., J. Gen.Virol. 72:1715-1720 (1991), incorporated herein by reference), mumpsvirus (Elango et al., J. Virol. 63:1413-1415 (1989), incorporated hereinby reference), and turkey rhinotracheitis virus (Ling et al., J. Gen.Virol. 73:1709-1715 (1992), incorporated herein by reference). The smallhydrophobic VP24 protein of filoviruses is thought to be a surfaceprotein (Bukreyev et al., Biochem. Mol. Biol. Int. 35:605-613 (1995),incorporated herein by reference) and is also a putative counterpart ofthe paramyxovirus SH protein.

The function of the SH protein has not been heretofore defined. In afusion assay in cells expressing plasmid-encoded proteins, efficientfusion of CV-1 cells by RSV proteins required the coexpression of the F,G and SH proteins (Heminway et al., Virology 200:801-805 (1994),incorporated herein by reference). Without wishing to be bound bytheory, several functions of the RSV SH protein may exist: (i) it mayenhance viral attachment or penetration (Heminway et al., supra.); (ii)it may be involved in virion morphogenesis; or (iii) it may have a“luxury” function distinct from a direct role in virus growth, such asinteraction with components of the host immune system as recentlydescribed for the V protein of Sendai virus (Kato et al., EMBO J.16:178-587 (1997), incorporated herein by reference). Function (i) or(ii) above may involve an activity that modifies membrane permeability,as has been suggested by others for some hydrophobic proteins of variousviruses (Maramorosh et al. (eds.), Advances in Virus Research 45:61-112(1995); Schubert et al., FEBS Lett. (1996); Lamb et al., Virology229:1-11 (1997), each incorporated herein by reference). All of thesepotential activities of the SH protein may be incorporated within theinvention according to the is methods and strategies described herein,to yield additional advantages in RSV recombinant vaccines.

To produce a recombinant RSV having a selected disruption of SH genefunction, the SH gene was deleted in its entirety from a parental RSVclone. The above described plasmid D46 plasmid is one such clone whichencodes a complete antigenomic RNA of strain A2 of RSV, which was usedsuccessfully to recover recombinant RSV (See U.S. patent applicationSer. No. 08/720/132; U.S. Provisional Patent Application No. 60/007,083,each incorporated herein by reference). This antigenome is one nt longerthan the naturally-occurring genome and contains several optionalrestriction site markers. The D46 plasmid was modified so that thecomplete SH gene was deleted, yielding plasmid D46/6368 (FIG. 7).

The construction of plasmid D46/6368 involved two parental subclones,D50, which contains a T7 promoter attached to the left-hand end of thegenome encompassing the leader region to the beginning of the L gene,and D39, which contains the end of M2 and the L gene attached at thedownstream end to a hammerhead ribozyme and tandem T7 transcriptionterminators. The D50 plasmid was digested with ScaI (position 4189 inthe complete 15,223-nucleotide antigenome sequence) and PacI (position4623) and the resulting 435 bp fragment was replaced with a short DNAfragment constructed from two complementary oligonucleotides. In thisexemplary deletion, the 435-bp fragment located between the ScaI andPacI sites corresponds to the very downstream end of the M gene, its GEsignal, and the complete SH gene except for the last six nucleotides ofits GE sequence (FIG. 7). This was replaced with the two syntheticpartially complementary oligonucleotides,5′-ACTCAAATAAGTTAATAAAAAATATCCCGGGAT-3′ [SEQ ID NO: 3] (positive-sensestrand, the M GE sequence is underlined, Xmal site is shown in italics,and the ScaI half-site and PacI sticky end at the left and rightrespectively are shown in bold italics) and5′-CCCGGGATATTTTTTATTAACTTATTTGAGT-3′ [SEQ ID NO: 4] (negative-sensestrand). This cDNA, called D50/6368, was used to accept the BamHI-Mlulfragment of D39 containing the remainder of the antigenome. Thisresulted in the plasmid D46/6368 which encoded the complete antigenomeexcept for the deleted sequence, an antigenome that is 14,825 nt long,398 nt shorter than the antigenome encoding by the wild type plasmidD46. The sequence of the insert was confirmed by dideoxynucleotidesequence analysis. An XmaI site was optionally introduced so thatinserts could easily be placed at this position in subsequent work.

Transfection, growth and passaging of virus, plaque purification, andantibody staining of viral plaques were generally performed according tothe procedures described hereinabove, but with two modifications: (i)cytosine arabinoside, an inhibitor of vaccinia virus was not used; (ii)HEp-2 cells used for transfection were incubated at either 32° C. or 37°C., and all recovered viruses were propagated at 37° C.

To evaluate total RNA and poly(A)+ RNA, cells were scraped andresuspended in 100 μl of water, and total intracellular RNA was isolatedusing Trizol™ reagent (Life Technologies) according the manufacturer'srecommendation (except that following isopropanol precipitation the RNAwas extracted twice with phenol-chloroform followed by ethanolprecipitation. Poly(A)+ RNA was isolated using the Oligotex mRNA kit(Qiagen, Chatsworth, Calif.).

To conduct reverse transcription and polymerase chain reaction (RT-PCR),the SH gene region was copied into cDNA and amplified. Totalintracellular RNA was subjected to reverse transcription withSuperscript II (Life Technologies) using as primer the positive sensesynthetic oligonucleotide 5-GAAAGTATATATTATGTT-3′ [SEQ ID NO: 5]. Thisprimer is complementary to nucleotides 3958-3975, which are upstream ofthe SH gene. An aliquot of the cDNA product was used as template in PCRusing as primer the above-mentioned oligonucleotide together with thenegative-sense oligonucleotide 5′-TATATAAGCACGATGATATG-3′ [SEQ ID NO:6].This latter primer corresponds to nucleotides 4763-4782 of the genome,which are downstream the SH gene. An initial 2 min. denaturation stepwas performed during which the Taq DNA polymerase was added, and then 33cycles were performed (denaturation, 1 min. at 94° C.; annealing, 1 min.at 39° C.; elongation, 2 min. at 72° C.). The products were thenanalyzed on a 2.5% agarose gel.

For Northern blot hybridization, RNA was separated by electrophoresis onagarose gels in the presence of formaldehyde and blotted tonitrocellulose. The blots were hybridized with [³²P]-CTP-labeled DNAprobes of the M, SH, G, F, M2 and L genes which were synthesizedindividually in vitro from cDNAs by Klenow polymerase with randompriming using synthetic hexamers (Boehringer Mannheim, Indianapolis,Ind.). Hybridized radioactivity was quantitated using the MolecularDynamics (Sunnyvale, Calif.) Phosphorlmager 445 SI.

³⁵S methionine labeling, immunoprecipitation, and polyacrylamide gelelectrophoresis procedures were performed as described previously(Bukreyev et al., supra). Electrophoresis was conducted using pre-cast4%-20% Tris-glycine gels (Novex).

For In vitro growth analysis, HEp-2, 293, CV-1, Vero, MRC-5, Africangreen monkey kidney (AGMK), bovine turbinate (BT), and MDBK cellmonolayers were used in a single-step growth cycle analysis. For eachtype of cells, three 25-cm² culture flasks were infected with 10⁷ PFU ofthe D46/6368 (SH-minus) or D46 (wild type recombinant) virus. Opti-MEM(Life Technologies) with 2% fetal bovine serum (FBS) (Summit) was usedfor HEp-2, Vero, 293, BT, MRC-5, and AGMK cells; E-MEM (LifeTechnologies) with 1%, or 2% FBS was used for MDBK or BT cells,respectively. After 3 hours adsorption at 37° C., cells were washed with4 ml medium three times each, 4 ml medium was added, and the cells wereincubated at 37° C. with 5% C0₂. Then, at various times afterinoculation (see below), 200 μl aliquots of medium supernatant wereremoved, adjusted to contain 100 mM magnesium sulfate and 50 mM HEPESbuffer (pH 7.5), flash-frozen and stored at −70° C. until titration;each aliquot taken was replaced with an equal amount of fresh medium.For titration, HEp-2 cells (24-well plates) were infected with 10-folddilutions of aliquots, and overlaid with Opti-MEM containing 2% FBS and0.9% methylcellulose (MCB Reagents). After incubation for 7 days, themedium was removed and the cell monolayer was fixed with 80% methanol at4° C. The plaques were incubated with a mixture of three monoclonalantibodies specific to RSV F protein, followed by goat anti-mouse IgGconjugated with horseradish peroxidase (Murphy et al., Vaccine 8:497-502(1990), incorporated herein by reference).

Recovery of infectious, recombinant RSV lacking SH gene functionfollowed the above described procedures, wherein the D46/6368 plasmidwas cotransfected into HEp-2 cells together with plasmids encoding theN, P, L and M2 ORF1 proteins, and the cells were simultaneously infectedwith a recombinant of the MVA strain of vaccinia virus that expresses T7RNA polymerase. Parallel cultures were transfected with the D46 wildtype cDNA under the same conditions. Medium supernatants were harvestedthree days post-transfection and passaged once. After 6 days incubationat 37° C., an aliquot of medium supernatant representing each originaltransfection was plated onto fresh HEp-2 cells, incubated for six daysunder methylcellulose overlay, and stained by reaction with a mixture ofthree monoclonal antibodies specific to the RSV F protein followed by asecond antibody conjugated with horseradish peroxidase. Morphology ofplaques of wild type recombinant virus versus the SH-minus virus werevery similar, except that the latter plaques were larger. Notably,plaques formed by each of the control and SH minus viruses containedsyncytia.

To confirm the absence of the SH gene in the genome of recoveredD46/6368 virus, cells were infected with the first passage of wild typeor SH-minus recombinant virus, and total intracellular RNA was recoveredand analyzed by RT-PCR. RT was performed with a positive-sense primerthat annealed upstream of the SH gene, at genome positions 3958-3975.PCR was performed using the same primer together with a negative-senseprimer representing nucleotides 4763-4782, downstream of the SH gene. Asshown in FIG. 8, wild type D46 virus yielded a single PCR productcorresponding to the predicted 824 bp fragment between positions 3958and 4782 (lane 3), In the case of the D46/6368 virus, the PCR productwas shorter and corresponded to the predicted 426 bp fragment containingthe deletion (lane 2). The generation of The PCR products was dependenton the RT step, showing that they were derived from RNA rather than DNA,as expected. Thus, RT-PCR analysis demonstrates that the genome ofD46/6368 virus contains the expected 398-nucleotide deletion at the SHlocus.

To examine the transcription of genes located upstream and downstreamthe SH gene, poly(A)+ mRNA was isolated from cells infected with the D46or D46/6368 virus and analyzed by Northern blot hybridization (FIG. 9).The intracellular RNA purposefully was not denatured prior to poly(A)+selection; thus, as shown below, the selected mRNA also containedgenomic RNA due to sandwich hybridization to mRNA. This permittedsimultaneous analysis of mRNA and genome, and the ability to relate theabundance of each mRNA to genomic RNA contained in the same gel lanemade it possible to compare mRNA abundance between lanes. The blots werehybridized with [³²P]-labeled DNA probes which were synthesized fromcDNA clones by random priming and thus contained probes of bothpolarities. Selected probes represented individually the M, SH, G, F,M2, and L genes (FIG. 9).

The SH probe hybridized to both subgenomic SH mRNA and genomic RNA inthe case of the wild type D46 virus but not for the D46/6368 virus (FIG.9). The probes specific for other RSV genes hybridized in each case tothe genome and to the expected major monocistronic mRNA for bothviruses. In addition, a number of previously described dicistronicreadthrough mRNAs also were detected with both viruses, such as theF-M2, G-F and P-M mRNAs.

The G-specific probe hybridized to an novel species specific to theD46/6368 virus, which appeared to be a readthrough of the M and G genes.This combination was possible due to the deletion of the intervening SHgene. The same species, specific to D46/6468 but not D46, appeared tohybridize in addition only with the M-specific probe.

Relative levels of synthesis were subsequently quantified for each mRNAof D46 versus D46/6368. For each pairwise comparison, the amount of mRNAin a given gel lane was normalized relative to the amount of genome.This comparison showed D46/6368 versus D46 expressed the following mRNAsin the indicated ratio (D46/6368 to D46): M (1.1), G (1.3), F (0.61), M2(0.32), and L (0.17).

To compare viral proteins synthesized by D46 versus D46/6368 RSV clones,HEp-2 cells were infected at an input multiplicity of infection of 2 PFUper cell and labeled by incubation with [³⁵S]methionine from 16 to 20 hpost-infection. Cell lysates were prepared and analyzed directly orfollowing immunoprecipitation using a rabbit antiserum raised againstpurified RSV virions. Total and immunoprecipitated proteins weresubjected to PAGE on 4-20% gradient gels (FIG. 10).

In the case of the D46 virus, the pattern of immunoprecipitated proteinsincluded the unglycosylated form of SH protein, SH0, and theN-glycosylated form, SHg, whereas neither species was evident for theD46/6368 virus (FIG. 10). The SH0 protein also could be detected in thepattern of total infected-cell proteins in the case of D46, but notD46/6368. Otherwise, the patterns of proteins synthesized by D46 versusD46/6368 were essentially indistinguishable. Phosphorimager analysis ofthe N, P, M, F₁, and M2 proteins in the pattern of immunoprecipitatedproteins showed that equivalent amounts were made by both viruses (FIG.10).

As described above, preliminary comparison of the D46 and D46/6368viruses by plaque assay indicated a difference in growth in vitro.Therefore, we further compared the two viruses with regard to plaquesize in HEp-2 cells. Particular care was taken to ensure that themonolayers were young and not overgrown, since these variables caneffect plaque size. After 7 days incubation at 37° C. undermethylcellulose, the monolayers were fixed with methanol andphotographed. This revealed a striking difference in plaque size.Measurement of 30 plaques of each virus viruses showed that the plaquesof the D46/6368 virus were on average 70% larger than those of the D46virus.

Further experiments were undertaken to render a growth curve analysisfor eight different cell lines representing different species anddifferent tissue origins, to compare efficiencies of replication of theD46 and D46/6368 viruses (Table 42). Triplicate monolayers of each typeof cell were infected with either virus, and samples were taken at 12 or24 hours intervals, and quantitated by plaque assay and antibodystaining. Surprisingly, the D46/6368 virus grew to higher titersrelative to the D46 wild type in three cell lines, namely HEp-2, 293 andAGMK-21 cells. In HEp-2 cells (FIG. 11), the titer of progeny D46/6368virus at 36 hours post infection was 2.6 fold greater than for the D46virus. In 293 cells (FIG. 12), the yield of D46/6368 virus was two foldgreater than that of the D46 virus at 36 hours post infection, and thisdifference increased to 4.8 and 12.6-fold at 60 and 84 h post infection,respectively. In AGMK-21 cells (FIG. 13), the yield of D46/6368 viruswas 3.2 times at 36 hours post infection. In MRC-5, Vero, CV-1, MDBK andBT cells, a significant difference in replication among mutant andwild-type virus was not observed, indicating that the growth of SH-minusRSV was not substantially affected by host range effects. TABLE 42Replication of D46/6368 Virus as Compared to D46 Virus in Various CellLines D46/6368 Virus Replication As Cell Type Host Tissue Type Comparedto D46 HEp-2 human larynx increased 293 human kidney increased MRC-5human lung similar Vero monkey kidney similar AGMK-21 monkey kidneyincreased CV-1 monkey kidney similar BT bovine turbinate similar MDBKbovine kidney similar

These and other findings herein demonstrate that deletion of the SH geneyields not only recoverable, infectious RSV, but a recombinant RSV whichexhibits substantially improved growth in tissue culture, based on bothyield of infectious virus and on plaque size. This improved growth intissue culture specified by the SH deletion provides useful tools fordeveloping RSV vaccines, for example by overcoming problems of poor RSVyields in culture. Moreover, these deletions are highly stable againstgenetic reversion, rendering RSV clones derived therefrom particularlyuseful as vaccine agents.

To evaluate replication, immunogenicity and protective efficacy of theexemplary SH-deletion clone in mice, respiratory-pathogen-free 13-weekold BALB/c mice in groups of 24 were inoculated intranasally under lightmethoxyflurane anesthesia on day 0 with 10⁶ PFU per animal in a 0.1 mlinoculum of wild type recombinant D46 virus, SH-minus recombinantD46/6368 virus, or biologically-derived cold-passaged (cp)temperature-sensitive (ts) virus cpts248/404 (Firestone et al., supra,(1996), incorporated herein by reference). This latter virus has beenextensively characterized in rodents, chimpanzees and humans, and ishighly restricted in replication in the upper and lower respiratorytract of the mouse. At each of days 4, 5, 6 and 8 post-inoculation, sixmice from each group were sacrificed by CO₂ asphyxiation, and nasalturbinates and lung tissue were obtained separately, homogenized, andused in plaque assay for quantitation of virus using the antibodystaining procedure described above.

In the upper respiratory tract, the SH minus D46/6368 virus exhibited anattenuation phenotype (FIG. 14). In the present example, its level ofreplication was 10-fold lower than that of the wild type virus, and wascomparable to that of the cpts248/404 virus. In contrast, in the lowerrespiratory tract (FIG. 15) the level of replication of the D46/6368virus was very similar to that of the wild type, whereas the cpts248/404virus was highly restricted. In additional studies, it was determinedthat the recombinant virus encoded by the parental D46 cDNA has a wildtype phenotype with regard to replication and virulence in naivechimpanzees, a fully permissive experimental animal. This indicates thatthe parental recombinant virus, assembled as it was from a laboratorystrain, contains a full complement of intact genes necessary forreplication and virulence in a permissive host. These and otherproperties of exemplary gene deletion RSV strains render a host ofmethods and strains available for vaccine use.

To further evaluate immunogenicity and protective efficacy ofrecombinant RSV, four additional groups of mice were inoculated asdescribed above with wild type recombinant, its SH-minus derivative, orthe cpts248/404 virus, or were mock infected. Four weeks later the micewere anesthetized, serum samples were taken, and a challenge inoculationof 10⁶ PFU of biologically-derived RSV strain A2 per animal wasadministered intranasally. Four days later the animals were sacrificedand nasal turbinates and lung tissues were harvested and assayed forinfectious RSV as described above. Serum IgG antibodies which bind tothe RSV F protein were quantitated in an ELISA using F glycoproteinwhich had been immunoaffinity-purified from RSV Long strain infectedcells (Murphy et al., supra, 1990).

The immunogenicity assays for the D46 wild type, D46/6368 SH-minus, andcpts248/404 viruses showed that mice which had been infected on day 0with any of the three viruses developed high levels of F-specific serumantibodies (Table 43). All of these test hosts were highly resistant inboth the upper and lower respiratory tracts to replication of RSVchallenge virus. Thus, the SH-minus mutant could not be distinguishedfrom its wild type parent on the basis of its ability to induceRSV-specific serum antibodies and protection in the mouse. TABLE 43 TheRSV D46/6368 SH-minus virus is immunogenic and protects the upper andlower respiratory tract of mice against wild-type challenge RSV A2Replication after challenge³ Serum antibody titer² (mean log₁₀pfu/gtissue) Immunizing No. of (reciprocal mean log₂) Nasal Virus¹ Mice Day 0Day 28 turbinates Lungs D46⁴ 6 7.3 15.0 <2.0 <1.7 D46/6368⁵ 6 7.3 15.02.1 <1.7 cpts248/404 6 7.0 12.6 2.3 <1.7 none 6 7.6 7.3 4.6 5.1¹Groups of BALB/c mice were immunized intranasally with 10⁶ pfu of theindicated virus on day 0.²Serum IgG antibody response was quantitated in an ELISA usingimmunopurified F glycoprotein from RSV subgroup A.³Mice were intranasally administered 10⁶ pfu of RSV A2 on day 28 andsacrificed 4 days later.⁴Wild type recombinant virus.⁵SH-minus recombinant virus.

Comparisons of the SH genes of different RSVs and differentpneumoviruses provide additional tools and methods for generating usefulRSV recombinant vaccines. For example, the two RSV antigenic subgroups,A and B, exhibit a relatively high degree of conservation in certain SHdomains. In two such domains, the N-terminal region and putativemembrane-spanning domains of RSV A and B display 84% identity at theamino acid level, while the C-terminal putative ectodomains are moredivergent (approx. 50% identity) (Collins et al., supra, 1990).Comparison of the SH genes of two human RSV subgroup B strains, 8/60 and18537, identified only a single amino acid difference (Anderson et al.,supra). The SH proteins of human versus bovine RSV are approximately 40%identical, and share major structural features including (i) anasymmetric distribution of conserved residues similar to that describedabove; (ii) very similar hydrophobicity profiles; (iii) the presence oftwo N-linked glycosylation sites with one site being on each side of thehydrophobic region; and (iv) a single cysteine residue on thecarboxyterininal side of the central hydrophobic region of each SHprotein. (Anderson et al., supra). By evaluating these and othersequence similarities and differences, selections can be made ofheterologous sequence(s) that can be substituted or inserted withininfectious RSV clones, for example to yield vaccines havingmulti-specific immunogenic effects.

Transcriptional analyses for D46 and D46/6368 yielded other importantfindings within the present example. Overall transcription levels weresubstantially the same for both viruses, whereas, Northern blot analysisrevealed certain differences in accumulation of individual mRNA species.Notably, transcription of the M gene, located upstream of the SH gene,was substantially the same for the two viruses. However, deletion of theSH gene resulted in increased transcription for its downstream neighbor,the G gene (FIG. 16). Based on these results, transcriptional levels ofselected RSV genes can be modulated simply by changing the respectivepolarities or proximity of genes on the RSV map. For example, the D46virus G gene is seventh in the gene order, whereas deletion of the SHgene places it in the sixth position, resulting in increased levels oftranscription. The observed increase in transcription associated withthis change in gene order was less than the 2.5 to 3-fold increase whichhas been reported in other recombinant viral systems (see, e.g., Kuo etal., supra, (1996)).

Another important finding revealed in the foregoing studies was thateach of the genes downstream of the G gene (F, M2 and L) was expressedless efficiently in the SH-minus mutant, and there was a steepergradient of polarity for D46/6368 versus the wild type D46 virusexhibited by these downstream genes (FIG. 16). Notably, the engineeredM-G intergenic region of D46/6368 that was left following the SHdeletion was 65 nt in length. In comparison, the longestnaturally-occurring intergenic regions in strain A2 are the 44-nt M-SH,46-nt F-M2, and 52-nt G-F intergenic regions, and strain 18537 has aF-M2 intergenic region of 56 nt (Johnson et al., J. Gen. Virol.69:2901-2906 (1988), incorporated herein by reference). Thenaturally-occurring intergenic regions of strain A2, which range in sizefrom one to 52 nt, did not substantially differ with regard to theireffect on transcriptional readthrough and polarity in a dicistronicminigenome (Kuo et al, supra, (1996)). However, testing of regionslonger than the 52-nt G-F region according to the methods of theinvention may establish an upper limit after which the polymerase isaffected more severely. Thus, the lower-than-expected increase in G genetranscription resulting from the change in gene order observed in theSH-minus deletion clones, as well as the reduced transcription of thedownstream genes, may be attributable to the greater length of theintergenic region that was engineered between M and G. In this regard,adjustment in the selected length of intergenic regions of RSV cloneswithin the invention is expected to provide yet additional tools andmethods for generating useful RSV vaccines.

The above findings offer two additional methods for altering levels ofRSV gene expression. First, the deletion of a nonessential gene canup-regulate the expression of downstream genes. Second, the insertion oflonger than wild-type intergenic regions provide methods for decreasingthe transcription of downstream genes. Decreased levels of expression ofdownstream genes are expected to specify attenuation phenotypes of therecombinant RSV in permissive hosts, e.g., chimpanzees and humans.

The finding that the SH-minus virus grows well in tissue culture andexhibits site-specific attenuation in the upper respiratory tractpresents novel advantages for vaccine development. Current RSV strainsunder evaluation as live virus vaccines contain, e.g., cp mutations(acquired during extensive passage at progressively lower temperatures;to yield cpRSV strains). Exemplary cp mutations involve five amino acidsubstitutions in N, F and L, and do not confer temperature-sensitivityor cold-adaptation. These mutations further do not significantly affectgrowth in tissue culture. They are host range mutations, because theyrestrict replication in the respiratory tract of chimpanzees and humansapproximately 100-fold in the lower respiratory tract. Another exemplarytype of mutation, ts mutations, has been acquired by chemicalmutagenesis of cpRSV. This type of mutation tends to preferentiallyrestrict virus replication in the lower respiratory tract, due to thegradient of increasing body temperature from the upper to the lowerrespiratory tract. In contrast to these cp and ts mutants, the SH-minusmutants described herein have distinct phenotypes of greater restrictionin the upper respiratory tract. This is particularly desirable forvaccine viruses for use in very young infants, because restriction ofreplication in the upper respiratory tract is required to ensure safevaccine administration in this vulnerable age group whose members breathpredominantly through the nose. Further, in any age group, reducedreplication in the upper respiratory tract will reduce morbidity fromotitis media. In addition to these advantages, the nature of SH deletionmutations, involving e.g., nearly 400 nt and ablation of an entire mRNA,represents a type of mutation which will be highly refractory toreversion.

EXAMPLE XIV Recombinant RSV Having a Deletion of the SH Gene Without theIntroduction of Heterologous Sequence

This example describes the production of a recombinant RSV in whichexpression of the SH protein has been ablated by removing apolynucleotide sequence encoding the SH protein, without introducingheterologous sequence as was done in the preceding Example XIII. In thatexample, an RSV clone D46/6368 in which the SH coding sequence had beenremoved also featured an M-G intergenic region that was extended to 65nucleotides in length compared to 52 nucleotides for the longestnaturally occurring strain A2 intergenic region. Also, this engineeredintergenic region contained heterologous sequence including a SmaI site.These changes yield, certain advantageous results, such as reducing theefficiency of transcription of downstream genes. However, for otherapplications it is desirable to make deletions or changes which are notaccompanied by the introduction of heterologous sequence, as illustratedin the present Example (FIG. 17).

The D13 plasmid, described hereinabove (see Example VII), contains theT7 promoter attached to the left-hand end of the genome encompassing theleader region, and the NS1, NS2, N, P, M, and SH genes (sequencepositions 1 to 4623 in the complete 15,223 antigenome sequence). TheScaI (position 4189) to PacI (position 4623) fragment was replaced withthe two partially-complementary synthetic oligonucleotides:ACTCAAATAAGTTAAT [SEQ ID NO:7] (positive-sense strand, the ScaI halfsite is italicized to the left and the PacI sticky end is italicized tothe right, and part of the GE signal is underlined), and TAACTTATTTGAGT[SEQ ID NO:8] (negative sense, the ScaI half-site is italicized on theright, and part of the GE signal is underlined). This mutation resultedin the deletion of the M GE signal, the M-SH intergenic region and thecomplete SH gene, and had the effect of moving the SH GE signal up toreplace that of the M gene. No heterologous nucleotides were introduced.The resulting cDNA, called D13/6340, was ligated with the G-F-M2 cDNApiece (see Example VII) to yield D50/6340, which spans from the leaderregion to the beginning of the L gene.

The StuI-BamHI fragment of D50/6340 (spanning positions 5613 to 8501,including the F and M2 genes) was excised and replaced with theequivalent fragment of D50-COR#1, which is isogenic except that itcontains the two “HEK” changes to the F gene described hereinabove. Theresulting D50/6340HEK was used to accept the BamHI-MluI fragment ofD39sites#12, which contains the remainder of the antigenome cDNA and theflanking ribozyme and T7 transcription terminators (see Example IX).D39sites#12 is a version of D39 which contains the “sites” mutations(see Table 39). This resulted in plasmid D46/6340HEK, encoding anantigenome lacking the SH gene and containing the “HEK” and “sites”changes. The D46/6340 antigenome cDNA is 417 bp shorter than itsparental D46 cDNA. The sequence of the ScaI PacI synthetic insert wasconfirmed by dideoxynucleotide sequencing. The cDNA was then usedsuccessfully to recover recombinant virus by the procedures describedhereinabove. The recovered D46/6340 SH deletion virus resembled theD46/6368 SH deletion virus described in Example XIII on the basis of itsplaque phenotype and growth characteristics.

EXAMPLE XV Knock-Out of NS2 Protein Expression

This example illustrates ablation of synthesis of an RSV protein, NS2.The selected method for ablation in this instance was introduction ofstop codons into a translational open reading frame (ORF).

D13 is a cDNA representing the left hand end of the complete antigenomecDNA, including the T7 promoter, leader region, and the NS1, NS2, N, P,M and SH genes (sequence: positions 1 to 4623) (FIG. 18). TheAatII-AflII fragment of this cDNA, containing the T7 promoter and NS1and NS2 genes, was subcloned into a pGem vector and subjected tooligonucleotide-directed mutagenesis to introduce two translational stopcodons into the NS2 ORF together with an XhoI site that was silent atthe translational level and served as a marker (FIG. 18). The sequenceof the mutation was confirmed, and the AatII-AflII fragment was insertedinto D13, which was then ligated with the PacI-BamHI fragment containingthe G, F and M2 genes, to yield cDNA D50 containing the insertedmutations. This was then ligated with the insert of D39 to yield acomplete antigenome cDNA which was used to recover recombinant virus.The presence of the mutation in the recombinant RSV was confirmed bysequencing of RT-PCR products and by XhoI digestion. In addition, theabsence of synthesis of NS2 protein was confirmed by Western blotanalysis using a rabbit antiserum raised against a synthetic peptiderepresenting the C-terminus of NS2.

FIG. 19 shows growth curves comparing the NS2-knock-out virus withrecombinant wild type, using the methods described above for theSH-minus virus. These results demonstrate that the rate of release ofinfectious virus was reduced for the NS2-knock-out virus compared towild type. In addition, comparison of the plaques of the mutant and wildtype viruses showed that those of the NS2-knock-out were greatly reducedin size. This type of mutation can thus be incorporated within viablerecombinant RSV to yield altered phenotypes, in this case reduced rateof virus growth and reduced plaque size in vitro. These and otherknock-out methods and mutants will therefore provide for yet additionalrecombinant RSV vaccine agents, based on the known correlation betweenreduced plaque size in vitro and attenuation in vivo.

EXAMPLE XVI Modulation of RSV Phenotype by Alteration of Cis-ActingRegulatory Sequence Elements

This example illustrates modulation of growth properties of arecombinant RSV virus by altering cis-acting transcription signals ofexemplary genes, NS1 and NS2.

The subcloned AatII-AflII fragment of D13, representing the lefthand endof the genome, was subjected to oligonucleotide-directed mutagenesis tointroduce changes at the GE signals of the NS1 and NS2 genes (FIG. 20).The NS1 GE signal sustained a single nucleotide substitution, whereasthat of NS2 sustained three substitutions and one insertion. Thesechanges had the effect of altering each signal to be identical to thenaturally-occurring GE signal of the N gene.

These mutations were confirmed by dideoxynucleotide sequence analysis,and the AatII-flII fragment was replaced into D13, which in turn wastaken through the above described steps to construct a complete D53 DNAcontaining the mutations. This clone was used to recover recombinantvirus.

The GE-mutant virus was analyzed in HEp-2 cells and compared to wildtype virus with respect to plaque size and growth curve. This showedthat the plaques were larger (on average 30% greater) than those of wildtype, and the rate of growth and yield of virus were increased. Theseresults are consistent with modification of gene expression by alteringcis-regulatory elements, for example to decrease levels of readthroughmRNAs and increase expression of proteins from downstream genes. Theresulting recombinant viruses will preferably exhibit increased growthkinetics and increased plaque size, providing but one example ofalteration of RSV phenotype by changing cis-acting regulatory elementsin the genome or antigenome. These and other examples herein demonstratea wide range of RSV mutations specific for phenotypic changes that areadvantageous for providing effective vaccine agents.

EXAMPLE XVII Recombinant RSV Having a Deletion of the NS1 Gene

This example describes the production of a recombinant RSV in whichexpression of the NS1 protein has been ablated by removal of thepolynucleotide sequence encoding the protein. The NS1 protein is a small139-amino acid species which is encoded by the first gene in the 3′ to5′ RSV gene map (Collins and Wertz, Proc. Natl. Acad. Sci. USA80:3208-3212 (1983), and Collins and Wertz, Virol. 243:442-451 (1985)).Its mRNA is the most abundant of the RSV mRNAs, consistent with thegeneral finding that there is a gradient of transcription such that theefficiency of gene expression is reduced with increasing distance fromthe promoter at the 3′ end. The NS1 protein is thought to be one of themost abundantly expressed RSV proteins, although a careful quantitativecomparison remains to be done. Despite its abundance, the function ofthe NS1 protein has not yet been clearly identified. In thereconstituted RSV minigenome system (Grosfeld et al., J. Virol.69:5677-5686 (1995), Collins et al., Proc. Natl. Acad. Sci. USA 93:81-85(1996)), in which transcription and RNA replication of a minigenome isdriven by viral proteins supplied by plasmids, the NS1 protein appearedto be a negative regulatory protein for both transcription and RNAreplication. Thus, it might be a regulatory protein. It is very possiblethat other functions exist which remain to be identified. The NS1protein does not have a known counterpart in other paramyxoviruses.Without wishing to be bound by theory, several functions of the NS1protein may exist: (i) it may be a viral regulatory factor as suggestedabove, (ii) it may be involved in some other aspect of the viral growthcycle, (iii) it may interact with the host cell, such as to preventapoptosis, and (iv) it may interact with the host immune system, such asto inhibit aspects of the host defense system such as interferonproduction, antigen processing or B or T cell functioning. All of thesepotential activities of the NS1 protein may yield additional advantagesin RSV recombinant vaccines.

To produce a recombinant RSV having a selected disruption of NS1 genefunction, the sequence encoding the NS1 protein was deleted in itsentirety from a parental RSV cDNA. In this exemplary deletion, thesubstrate for the mutagenesis reaction was plasmid D13, which wasdescribed hereinabove and contains the left hand end of the completeantigenome cDNA including the T7 RNA promoter, the leader region, andthe NS1, NS2, N, P, M, and SH genes (sequence positions 1 to 4623). Themutagenesis was done by the method of Byrappa et al. (Byrappa et al.,Genome Res. 5:404-407 (1995)), in which synthetic primers whichincorporate the desired change are made to face in opposite directionson the plasmid, which is then amplified by PCR, ligated, and transformedinto bacteria. The forward PCR primer was GACACAACCCACAATGATAATACACCAC[SEQ ID NO:9] (the second codon of the NS2 ORF is italicized), and thereverse PCR primer was CATCTCTAACCAAGGGAGTTAAATTTAAGTGG [SEQ ID NO:10](the complement to the initiation codon of the NS2 ORF is italicized).D13 was used as the template for PCR with a high-fidelity polymerase.The deletion was made to span from immediately upstream of the AUG startsite of the NS1 ORF to immediately upstream of the AUG start site of theNS2 ORF (FIG. 21). This resulted in the deletion of 529 bp including theNS1 coding sequence, the NS1 GE signal, the NS1-NS2 intergenic region,and the NS2 GS signal. It had the effect of fusing the upstream end ofthe NS1 gene, namely its GS and non-protein-coding region, to the NS2ORF. This part of the NS1 gene was retained expressly because it isimmediately adjacent to the leader region and thus might containsequences important in transcription or RNA replication. The regioncontaining the mutation was confirmed by sequence analysis. Then, the˜2230 bp segment between the Aat2 and Avr2 sites (FIG. 21) was excisedand inserted into a fresh copy of D13, a step that would therebypreclude the possibility of PCR error elsewhere in the RSV cDNA. The D13plasmid containing the deletion of NS1 (D13ΔNS1) was used to construct acomplete antigenome (D53ΔNS1) which contained the “HEK” and “sites”changes.

The D53ΔNS1 plasmid was then used to recover virus. Interestingly, therecovered RSVΔNS1 virus produced small plaques in tissue culture. Thepresence of the deletion was confirmed by RT-PCR. The fact that theRSVΔNS1 virus can grow, albeit with reduced efficiency, identifies theNS1 protein as an accessory protein, one that is dispensable to virusgrowth. The plaque size of the RSVΔNS1 virus was similar to that of theNS2-knock out virus described above in which expression of the NS2protein was ablated by the introduction of translational stop codonsinto its coding sequence (Example XV). The small plaque phenotype iscommonly associated with attenuating mutations. This type of mutationcan thus be incorporated within viable recombinant RSV to yield alteredphenotypes. These and other knock-out methods and mutants will thereforeprovide for yet additional recombinant RSV vaccine agents, based on theknown correlation between plaque size in vitro and attenuation in vivo.

EXAMPLE XVIII Recombinant RSV Having a Deletion of the NS2 Gene

This example describes the production of a recombinant RSV in whichexpression of the NS2 protein has been ablated by removal of apolynucleotide sequence encoding the protein. In Example XV, above, arecombinant virus (called NS2 knock-out or NS2-KO) was produced in whichexpression of the NS2 gene was ablated by the introduction of twotranslational stop codons into its coding sequence rather than bydeletion of the coding sequence. Ablation of expression of the NS2protein in the prototypic NS2-KO virus was associated with the smallplaque phenotype and reduced kinetics of virus growth in vitro.Subsequent analysis showed that, upon passage of NS2 KO, it was possibleto recover low levels of virus which had reverted to wild type growthcharacteristics. Sequence analysis showed that the two introducedtranslational stop codons had mutated into sense codons, albeit withcoding assignments different than in the parental wild type virus. Thiswas sufficient to restore synthesis of the NS2 protein as confirmed byWestern blot analysis, which accounted for the wild type phenotype. Thepresent strategy offers an improvement because the complete NS2 gene isremoved and thus same-site reversion cannot occur.

The NS2 protein is a small 124-amino acid protein which is encoded bythe second gene in the gene order (Collins and Wertz, Proc. Natl. Acad.Sci. USA 80:3208-3212 (1983), and Collins and Wertz, Virol. 243:442 451(1985). Its mRNA is the second most abundant of the RSV mRNAs, and theNS2 protein is thought to be one of the most abundantly-expressed RSVproteins, although a careful quantitative comparison remains to be done.Despite its abundance, the function of the NS2 protein remains to beidentified. In the reconstituted RSV minigenome system (Grosfeld et al.,J. Virol. 69:5677-5686 (1995), Collins et al., Proc. Natl. Acad. Sci.USA 93:81-85 (1996)), in which transcription and RNA replication of aminigenome is driven by viral proteins supplied by plasmids, the NS2protein had a modest negative regulatory effect against bothtranscription and RNA replication. Relatively high levels of NS2 proteinexpression were required to observe this effect, and so its significanceis unclear. Thus, as suggested for the NS1 protein, NS2 might: (i) be aviral regulatory factor, (ii) be involved in some other aspect of theviral growth cycle, (iii) interact with the host cell, such as toprevent apoptosis, and (iv) interact with the host defense system, suchas to inhibit aspects of the host immune system such as interferonproduction or aspects of antigen processing or B or T cell functioning.All of these potential activities of the NS2 protein may be incorporatedwithin the invention according to the methods and strategies describedherein, to yield additional advantages in RSV recombinant vaccines.

To produce a recombinant RSV having a selected disruption of NS2 genefunction, the sequence encoding its mRNA was deleted from a parental RSVcDNA. In this exemplary deletion, the substrate for the mutagenesisreaction was plasmid D13, which was mentioned previously and containsthe left hand end of the complete antigenome cDNA including the T7 RNApromoter, the leader region, and the NS1, NS2, N, P, M, and SH genes(sequence positions 1 to 4623). The mutagenesis was done by the methodof Byrappa et al. (Byrappa et al., Genome Res. 5:404-407 (1995)), inwhich synthetic primers which incorporate the desired change are made toface in opposite directions on the plasmid, which is then amplified byPCR, ligated, and transformed into bacteria. The forward PCR primer wasTTAAGGAGAGATATAAGATAGAAGATG [SEQ ID NO:11] (sequence from the NS2 Nintergenic region is underlined, and the first nucleotide of the N GSsignal is italicized), and the reverse PCR primer wasGTTTTATATTAACTAATGGTGTTAGTG [SEQ ID NO:12] (the complement to the NS1 GEsignal is underlined). D13 was used as the template for PCR with ahigh-fidelity polymerase. The deletion was made to span from immediatelydownstream of the NS1 GE signal to immediately downstream of the NS2 GSsignal (FIG. 22). Thus, the mutation deleted 522 nucleotides includingthe NS1-NS2 intergenic region and the complete NS2 gene (FIG. 22).

In the D13 plasmid containing the deletion, the region of the mutationwas confirmed by sequence analysis. Then, the ˜2230 bp segment betweenthe Aat2 and Avr2 sites (FIG. 22) was excised and inserted into a freshcopy of D13, a step that would thereby preclude the possibility of PCRerror elsewhere in the RSV cDNA. The D13 plasmid containing the deletionof NS2 (D13ΔNS2) was used to construct a complete antigenome (D53ΔNS2)which contained all of the “sites” and “HEK” changes.

The D53ΔNS2 plasmid was then used to recover virus. As is describedabove for the NS2-KO virus (see Example XV), the RSVΔNS2 produced smallplaques. The presence of the deletion was confirmed by RT-PCR. The factthat the RSVΔNS2 virus can grow, albeit with reduced efficiency,identified the NS2 protein as an accessory protein, one that isdispensable to virus growth. The small plaque phenotype is commonlyassociated with attenuating mutations and can thus be incorporatedwithin viable recombinant RSV to yield altered phenotypes. These andother knock-out methods and mutants will therefore provide for yetadditional recombinant RSV vaccine agents, based on the knowncorrelation between plaque size in vitro and attenuation in vivo.

EXAMPLE XIX Ablation of the Translational Start Site for the SecretedForm of the G Glycoprotein

This example describes the production of a recombinant RSV in which thetranslational start site for the secreted form of the G glycoprotein hasbeen ablated. The RSV G protein is synthesized in two forms: as ananchored type II integral membrane protein and as a N terminallyresected form which lacks essentially all of the membrane anchor and issecreted (Hendricks et al., J. Virol. 62:2228-2233 (1988)). The twoforms have been shown to be derived by translational initiation at twodifferent start sites: the longer form initiates at the first AUG of theG ORF, and the second initiates at the second AUG of the ORF at codon 48and is further processed by proteolysis (Roberts et al., J. Virol. 68:4538-4546 (1994)). The presence of this second start site is highlyconserved, being present in all strains of human, bovine and ovine RSVsequenced to date. It has been suggested that the soluble form of the Gprotein-might mitigate host immunity by acting as a decoy to trapneutralizing antibodies. Also, soluble G has been implicated inpreferential stimulation of a Th2-biased response, which in turn appearsto be associated with enhanced immunopathology upon subsequent exposureto RSV. With regard to an RSV vaccine virus, it would be highlydesirable to minimize antibody trapping or imbalanced stimulation of theimmune system, and so it would be desirable to ablate expression of thesecreted form of the G protein. This would represent a type of mutationwhose action would be to qualitatively and/or quantitatively alter thehost immune response, rather than to directly attenuate the virus.

Plasmid pUC19 bearing the G, F and M2 genes (see Example VII) was usedas template in PCR mutagenesis by the procedure of Byrappa et al.(Byrappa et al., Genome Res. 5:404-407 (1995). The forward PCR primerwas: TTATAATTGCAGCCATCATATTCATAGCCTCGG [SEQ ID NO:13], and the reverseprimer was: GTGAAGTTGAGATTACAATTGCCAGAATGG [SEQ ID NO: 14] (thecomplement of the two nucleotide changes is underlined). This resultedin two amino acid coding changes (see FIG. 23), namely AUG-48 to AUU-48,which ablates the translational start site, and AUA-49 to GUA-49, whichcontributes to the insertion of an MfeI site for the purpose ofmonitoring the mutation.

The sequence surrounding the site of mutation was confirmed bydideoxynucleotide sequencing. Then, the PacI-StuI fragment, whichcontains the G gene, was substituted into plasmid D50, which isdescribed hereinabove and contains the first nine genes from the leaderto the beginning of the L gene. This was then used to construct acomplete antigenome cDNA, D53/GM48I, which was used to recover virus.These mutations have previously been shown to ablate the expression ofthe secreted form of G under conditions where the G cDNA was expressedin isolation from the other RSV genes by a recombinant vaccinia virus(Roberts et al., J. Virol. 68:4538-4546 (1994)).

Two isolates of the recovered D53/M48I virus were evaluated for growthkinetics in vitro in parallel with wild type recombinant RSV (FIG. 24).This showed that both viruses grew somewhat more slowly, and to somewhatlower titers, than did the wild type. This difference in growth might bedue to the reduced expression of the G protein, which would be expectedto occur since the AUG-48 of the secreted form was eliminated whereas inthis Example the AUG-1 of the membrane-bound form was not modified toincrease its expression. In nature, the expression of AUG-1 of the G ORFis thought to be suboptimal because it is preceded in the G sequence byanother AUG in another reading frame which opens a short ORF thatoverlaps AUG-1 of the G ORF and thus would reduce its expression. Itmight be anticipated that additional modification of the antigenome cDNAto eliminate this ORF might restore full growth properties.Alternatively, the mutations at position 48 and 49, acting alone or inconcert, might be deleterious to the function of the G protein. Theamino acid at position 49 could be restored to its natural codingassignment, and a different amino acid substitution could be chosen atposition 48, which might restore full growth properties. Thesepossibilities can be readily evaluated with the methods and materialsdescribed here. Nonetheless, the recover of the D53/GM48I virus showsthat the translational start site for the secreted form can be ablated,and this virus is now available for evaluation in experimental animalsand humans.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1-62. (canceled)
 63. An infectious recombinant respiratory syncytialvirus (RSV) comprising a RSV genome or antigenome, a major nucleocapsid(N) protein, a nucleocapsid phosphoprotein (P), a large polymeraseprotein (L), and an M2ORF1 RNA polymerase elongation factor, wherein amodification is introduced within the genome or antigenome comprising adeletion, insertion, substitution, rearrangement, or nucleotidemodification of a cis-acting regulatory sequence within the recombinantRSV genome or antigenome, said recombinant RSV being attenuated by 10-to 1000-fold compared to wild type RSV in the respiratory tract of aprimate host.
 64. The recombinant RSV of claim 63, wherein thecis-acting regulatory sequence is a gene-start (GS) signal or a (GE)signal.
 65. The recombinant RSV of claim 64, wherein a GS or GE signalis deleted or inserted in the genome or antigenome.
 66. The recombinantRSV of claim 64, wherein a GS or GE signal is substituted in the genomeor antigenome by a heterologous GS or GE sequence.
 67. The recombinantRSV of claim 66, wherein the heterologous GS or GE sequence is of adifferent RSV gene.
 68. The recombinant RSV of claim 67, wherein a GEsignal of the RSV NS1 or NS2 gene is replaced by a corresponding GEsequence of the RSV N gene.
 69. The recombinant RSV of claim 66, whereinthe heterologous GS or GE sequence is of a heterologous negativestranded virus.
 70. The recombinant RSV of claim 69, wherein theheterologous GS or GE sequence is of a human RSV A or RSV B subgroup.71. The recombinant RSV of claim 69, wherein the heterologous GS or GEsequence is of a non-human RSV.
 72. The recombinant RSV of claim 70,wherein the heterologous GS or GE sequence is of a bovine RSV.
 73. Therecombinant RSV of claim 70, wherein the heterologous GS or GE sequenceis of a parainfluenza virus (PIV).
 74. The recombinant RSV of claim 73,wherein the heterologous GS or GE sequence is of a PIV3 virus.
 75. Therecombinant RSV of claim 64, wherein a nucleotide sequence of agene-start (GS) or gene-end (GE) signal is altered in the genome orantigenome.
 76. The recombinant RSV of claim 64, wherein a gene-start(GS) or gene-end (GE) signal is rearranged by changing a position of the(GS) or gene-end (GE) signal in the recombinant genome or antigenome.77. The recombinant RSV of claim 63, wherein a modification isintroduced within the recombinant genome or antigenome comprising apartial or complete gene deletion, a change in gene position, or one ormore nucleotide change(s) that modulate expression of a selected gene.78. The recombinant RSV of claim 77, wherein a RSV gene is deleted inwhole or in part.
 79. The recombinant RSV of claim 78, wherein a SH,NS1, NS2, or G gene is deleted in whole or in part.
 80. The recombinantRSV of claim 77, wherein expression of a selected RSV gene is reduced orablated by introduction of one or more translation termination codons.81. The recombinant RSV of claim 80, wherein expression of a selectedRSV gene is reduced or ablated by introduction of multiple translationtermination codons.
 82. The recombinant RSV of claim 77, whereinexpression of a selected RSV gene is reduced or ablated by introductionof a frame shift mutation in the gene.
 83. The recombinant RSV of claim77, wherein expression of a selected RSV gene is modulated byintroduction, modification or ablation of a translational start sitewithin the gene.
 84. The recombinant RSV of claim 77, wherein a positionof one or more gene(s) in the recombinant genome or antigenome isaltered relative to a RSV promoter.
 85. The recombinant RSV of claim 84,wherein said position of said one or more gene(s) is changed to a morepromoter-proximal or promoter-distal location by deletion or insertionof a coding or non-coding polynucleotide sequence within the recombinantgenome or antigenome upstream of said one or more gene(s).
 86. Therecombinant RSV of claim 84, wherein positions of multiple genes in therecombinant genome or antigenome are altered by changing their relativegene order.
 87. The recombinant RSV of claim 63, wherein the recombinantgenome or antigenome is further modified to incorporate one or moreattenuating mutation(s) present in one or more biologically derivedmutant human RSV strain(s).
 88. The recombinant RSV of claim 87, whereinthe recombinant genome or antigenome is further modified to incorporateat least one and up to a full complement of attenuating mutationspresent within a panel of biologically derived mutant human RSV strains,said panel comprising cpts RSV 248 (ATCC VR 2450), cpts RSV 248/404(ATCC VR 2454), cpts RSV 248/955 (ATCC VR 2453), cpts RSV 530 (ATCC VR2452), cpts RSV 530/1009 (ATCC VR 2451), cpts RSV 530/1030 (ATCC VR2455), RSV B-1 cp52/2B5 (ATCC VR 2542), and RSV B-1 cp-23 (ATCC VR2579).
 89. The recombinant RSV of claim 87, wherein the recombinantgenome or antigenome is further modified to incorporate at least one andup to a full complement of attenuating mutations specifying an aminoacid substitution at Val267 in the RSV N gene, Glu218 and/or Thr523 inthe RSV F gene, Cys319 Phe 521, Gln831, Met1169, Tyr1321 and/or His 1690in the RSV polymerase gene L, and a nucleotide substitution in thegene-start sequence of gene M2.
 90. The recombinant RSV of claim 87,wherein the recombinant genome or antigenome incorporates at least twoattenuating mutations.
 91. The recombinant RSV of claim 63, wherein therecombinant genome or antigenome comprises a partial or complete humanRSV genome or antigenome of one RSV subgroup or strain combined with aheterologous gene or gene segment from a different, human or non-humanRSV subgroup or strain to form a chimeric genome or antigenome.
 92. Therecombinant RSV of claim 91, wherein the heterologous gene or genesegment is from a human RSV subgroup A, human RSV subgroup B, bovineRSV, or murine RSV.
 93. The recombinant RSV of claim 91, wherein thechimeric genome or antigenome comprises a partial or complete human RSVA subgroup genome or antigenome combined with a heterologous gene orgene segment encoding a RSV F, G or SH glycoprotein or a cytoplasmicdomain, transmembrane domain, ectodomain or immunogenic epitope thereoffrom a human RSV B subgroup virus.
 94. The chimeric RSV of claim 93,wherein both human RSV B subgroup glycoprotein genes F and G aresubstituted to replace counterpart F and G glycoprotein genes in apartial RSV A genome or antigenome.
 95. The recombinant RSV of claim 93,wherein the chimeric genome or antigenome comprises a partial orcomplete human RSV B subgroup genome or antigenome combined with aheterologous gene or gene segment from a human RSV A subgroup virus. 96.The recombinant RSV of claim 91, wherein the chimeric genome orantigenome comprises a partial or complete RSV background genome orantigenome of a human or bovine RSV combined with a heterologous gene orgenome segment of a different RSV to form a human-bovine chimeric RSVgenome or antigenome.
 97. The recombinant RSV of claim 63, wherein therecombinant genome or antigenome incorporates a heterologous gene orgenome segment from parainfluenza virus (PIV).
 98. The recombinant RSVof claim 97, wherein the gene or genome segment encodes a PIV HN or Fglycoprotein or immunogenic domain or epitope thereof.
 99. Therecombinant RSV of claim 97, wherein the genome segment encodes one ormore immunogenic protein(s), protein domain(s) or epitope(s) HPIV1,HPfV2, and/or HPIV3.
 100. The recombinant RSV of claim 63, wherein therecombinant genome or antigenome is further modified to encode a non-RSVmolecule selected from a cytokine, a T-helper epitope, or a protein of amicrobial pathogen capable of eliciting a protective immune response ina mammalian host.
 101. The recombinant RSV of claim 63 which is acomplete virus.
 102. The recombinant RSV of claim 63 which is a subviralparticle.
 103. The recombinant RSV of claim 63, formulated in a dose of10³ to 10⁶ PFU of attenuated virus.
 104. A method for eliciting animmune response in a mammalian subject directed against respiratorysyncytial virus, which comprises administering to the subject animmunologically sufficient amount of the isolated attenuated recombinantRSV of claim
 63. 105. The method of claim 104, wherein the recombinantvirus is administered in a dose of 10³ to 10⁶ PFU of the attenuated RSV.106. The method of claim 104, wherein the recombinant virus isadministered to the upper respiratory tract.
 107. The method of claim106, wherein the recombinant virus is administered by spray, droplet oraerosol.
 108. The method of claim 104, wherein the recombinant virus isadministered to an individual seronegative for antibodies to RSV orpossessing transplacentally acquired maternal antibodies to RSV.
 109. Animmunogenic composition effective to elicit an immune response directedagainst RSV, which comprises an immunologically sufficient amount of theisolated attenuated recombinant RSV of claim 63 in a physiologicallyacceptable carrier.
 110. The immunogenic composition of claim 109,formulated in a dose of 10³ to 10⁶ PFU of the attenuated RSV.
 111. Theimmunogenic composition of claim 109, formulated for administration tothe upper respiratory tract by spray, droplet or aerosol.
 112. Theimmunogenic composition of claim 109, wherein the recombinant RSVelicits an immune response against human RSV A, human RSV B, or both.113. An expression vector comprising an isolated polynucleotide moleculeencoding a respiratory syncytial virus (RSV) genome or antigenomemodified by a deletion, insertion, substitution, rearrangement, ornucleotide modification of a cisacting regulatory sequence.
 114. Anisolated polynucleotide molecule comprising a respiratory syncytialvirus (RSV) genome or antigenome which is modified by a deletion,insertion, substitution, rearrangement, or nucleotide modification of acis-acting regulatory sequence, or by introduction of a translationtermination codon, said isolated polynucleotide providing 10-fold to1000-fold attenuation of replication in the respiratory tract of aprimate host of a RSV having a genome or antigenome comprising saidpolynucleotide.
 115. The isolated polynucleotide molecule of claim 114,wherein the cis-acting regulatory sequence is a gene-start (GS) signalor a (GE) signal.
 116. The isolated polynucleotide molecule of claim114, wherein the cis-acting regulatory sequence occurs within a 3′leader, 5′ trailer or intergenic region of the RSV genome or antigenome.117. The isolated polynucleotide molecule of claim 114, wherein thecis-acting regulatory sequence is a RSV promoter element.
 118. Theisolated polynucleotide molecule of claim 114, wherein a furthermodification is introduced within the recombinant genome or antigenomecomprising a partial or complete gene deletion, a change in geneposition, or one or more nucleotide change(s) that modulate expressionof a selected gene.
 119. The isolated polynucleotide molecule of claim118, wherein a RSV gene is deleted in whole or in part.
 120. Theisolated polynucleotide molecule of claim 114, wherein expression of aselected RSV gene is reduced or ablated by introduction of one or moretranslation termination codons in the recombinant genome or antigenome.121. The isolated polynucleotide molecule of claim 114, whereinexpression of a selected RSV gene is reduced or ablated by introductionof a frame shift mutation in the gene.
 122. The isolated polynucleotidemolecule of claim 114, wherein expression of a selected RSV gene ismodulated by introduction, modification or ablation of a translationalstart site within the gene.
 123. The isolated polynucleotide molecule ofclaim 114, wherein a position of one or more gene(s) in the recombinantgenome or antigenome is altered relative to a RSV promoter.
 124. Theisolated polynucleotide molecule of claim 114, wherein the recombinantgenome or antigenome is further modified to incorporate one or moreattenuating mutation(s) present in one or more biologically derivedmutant human RSV strain(s).
 125. The isolated polynucleotide molecule ofclaim 124, wherein the recombinant genome or antigenome is furthermodified to incorporate at least one and up to a full complement ofattenuating mutations specifying an amino acid substitution at Val 267in the RSV N gene, Glu218 and/or Thr523 in the RSV F gene, Cys319 Phe521, Gln831, Met1169, Tyr1321 and/or His 1690 in the RSV polymerase geneL, and a nucleotide substitution in the gene-start sequence of gene M2.126. The isolated polynucleotide molecule of claim 114, wherein therecombinant genome or antigenome comprises a partial or complete humanRSV genome or antigenome of one RSV subgroup or strain combined with aheterologous gene or gene segment from a different, human or non-humanRSV subgroup or strain to form a chimeric genome or antigenome.
 127. Theisolated polynucleotide molecule of claim 126, wherein the heterologousgene or gene segment is from a human RSV subgroup A, human RSV subgroupB, bovine RSV, or murine RSV.
 128. The isolated polynucleotide moleculeof claim 127, wherein the chimeric genome or antigenome comprises apartial or complete human RSV A subgroup genome or antigenome combinedwith a heterologous gene or gene segment encoding a RSV F, G or SHglycoprotein or a cytoplasmic domain, transmembrane domain, ectodomainor immunogenic epitope thereof from a human RSV B subgroup virus. 129.The isolated polynucleotide molecule of claim 128, wherein both humanRSV B subgroup glycoprotein genes F and G are substituted to replacecounterpart F and G glycoprotein genes in a partial RSV A genome orantigenome.
 130. The recombinant RSV of claim 114, wherein therecombinant genome or antigenome comprises a partial or complete RSVbackground genome or antigenome of a human or bovine RSV combined with aheterologous gene or genome segment of a different RSV to form ahuman-bovine chimeric RSV genome or antigenome.
 131. The isolatedpolynucleotide molecule of claim 114, wherein the recombinant genome orantigenome incorporates a heterologous gene or genome segment fromparainfluenza virus (PIV).
 132. The isolated polynucleotide molecule ofclaim 114, wherein the recombinant genome or antigenome is furthermodified to encode a non-RSV molecule selected from a cytokine, aT-helper epitope, or a protein of a microbial pathogen capable ofeliciting a protect immune response in a mammalian host.